CZ358399A3 - Methods and compositions for synthesis of poly-unsaturated fatty acids with long chains - Google Patents

Abstract

Desaturase fatty acids capable of catalyzing are described converting oleic acid to linolenic acid, acids linolenic to gamma-linolenic acid or alfalinolenic acid to stearidonic acid. It is further described nucleic acid sequences encoding desaturases, sequences the nucleic acids that hybridize with them, the DNA construct comprising a desaturase gene and a recombinant host a microorganism or an animal expressing an increased amount desaturase. BACKGROUND OF THE INVENTION The present invention relates to methods for desaturating fatty acid and desaturated fatty acid production by expression increased amount of desaturase. Fatty acids and oils containing desaturated desaturates are produced in recombinant host microorganisms or animals. Pharmaceutical compositions are also disclosed, baby food or food supplements containing greasy acid desaturated by desaturase recombinant host microorganisms or methods and compositions for synthesizing polyunsaturated fatty acids long chain acids.

Description

Methods and compositions for the synthesis of long chain polyunsaturated fatty acids.

Technical field

The invention describes the modulation steps of the long-enzymatic unsaturated fatty acid components unsaturated in a microorganism or animal.

enzymes and / or poly (PUFA) production)

BACKGROUND OF THE INVENTION

The two main groups of polyunsaturated fatty acids (PUFAs) are? 3-fatty acids, represented by icosapentaenoic acid (EPA), and? 6-fatty acids, which are arachidonic acid (ARA). PUFAs are important components of the plasma membrane of cells where they can exist in forms such as phospholipids. PUFAs are essential for proper development, especially in infant brain development, tissue formation and repair. PUFAs also serve as precursors to other molecules that are important to humans and animals, including prostacyclin, icosanoids, leukotrienes, and prostaglandins.

The four most important long-chain PUFAs include docosahexaenoic acid (DHA) and EPA, which are primarily found in different types of fish oil, and gana-linolenic acid (GLA), which is found in the seeds of a number of plants that include {Oenothera biennis ), borage (Borago officinahs) and blackcurrant (Ribes nigrum), and stearidonic acid (SDA), which is found in marine oils and in plant seeds. GLA and other important long-chain PUFAs such as arachidonic acid (ARA) are found in filamentous fungi. ARAs can be isolated from animal tissue including liver and adrenaline secreting glands. GLA, ARA, EPA and SDA are alone or as food precursors to important long-chain fatty acids involved in microorganisms including Phytium and Porphyridium.

prostaglandin synthesis, heart disease treatment, and brain tissue development.

In the case of DHA, there are a number of sources for commercial production, including various marine organisms, oils obtained from marine fish living in cold waters, and egg yolk fractions. In the case of ARA, the genus Mortierella, Entomophthora can be used for commercial production

Trichodesma and Echium. Commercial sources of GLA include primrose, blackcurrant and borage. However, there are several disadvantages associated with commercial production of PUFAs from natural sources. Natural PUFA sources, such as animals and plants, exhibit a highly heterogeneous oil composition. Oils obtained from these sources must be extensively purified in order to separate one or more of the required PUFAs or produce an oil that is enriched in one or more PUFAs. Natural resources are also exposed to uncontrolled availability fluctuations. Fish stocks may also be weakened by overfishing. Fish oil has an unpleasant taste and odor, the removal of which is not economically advantageous and such a product may not be acceptable as a dietary supplement. Animal oils and certain fish oils may accumulate in environmental pollutants. Here, animal oils and especially fish oils can accumulate. Variations in yield from fish and plant sources can affect weather and disease incidence. The land available for the production of alternative oil-producing crops compete with the expansion of the human population, which is associated with increasing food production needs on the remaining arable land. Crops that produce PUFA, such as borage, have not been adapted to large-scale cultivation and do not perform well when cultivated as a monoculture.

crops are then not economical compared to better established crops.

Large-scale fermentation of organisms such as Mortierella is also costly. Natural animal tissues contain small

Growing such thriving and • · ·

amount of ARA and difficult to process. Microorganisms such as Porphyridium and Mortieilera are difficult to cultivate on a commercial scale.

Dietary supplements and pharmaceutical formulations containing PUFAs have the same drawbacks as PUFA sources. Supplements, such as fish oil capsules, may contain a small amount of certain desired components and thus need to be applied in high doses. High doses lead to the digestion of large amounts of undesirable components, including pollutants. The unpleasant taste and odor of the supplements may make them undesirable and the patient may find it difficult to follow the treatment plan. Great care must be taken when administering fatty acid supplements. Excessive uptake can lead to suppression of endogenous biosynthetic pathways and to competition with other essential fatty acids in various lipid fractions in vivo, leading to undesirable results. For example, Eskimos whose diet contains large amounts of ω3 fatty acids have an increased tendency to vomit (US Patent No. 4,874,603).

Many enzymes are involved in PUFA biosynthesis. Linolenic acid (LA, 18: 2 Δ9, 12) is produced from oleic acid (18: 1 Δ °) by Á12-desaturase. Production of ARA (20: 4 ,5, 8, 11, 14) from dihomo-gamma-linoleic acid (DGLA, 20: 3 Δ8, 11, 14) is catalyzed by -5-desaturase. However, animals cannot desaturate beyond Δ9 and therefore cannot convert oleic acid (18: 1 Δ9) to linolenic acid (18: 2 Δ912). Animals cannot synthesize cc-noenoic acid (ALA, 18: 3 Δ9, 12, 15). Other eukaryotes including fungi and plants have enzymes that cause desaturation at the Δ12 and Δ15 positions. Therefore, the major polyunsaturated fatty acids of animals are obtained either from food and / or desaturation and elongation of linolenic acid (18: 2 Δ9, 12) or α-linolenic acid (18: 3 Δ9, 12, 15). Therefore, there is such an interest in the possibility of obtaining genetic material involved in PUFA biosynthesis from species that naturally produce fatty acids and express the isolated material in a microbial or animal system that can be used to produce a commercial amount of one. fatty acid desaturases, genes encoding them and recombinant methods for their production. Further, it is necessary to develop economical methods of producing specific PUFAs.

manipulated for or more PUFAs

Relevant literature

The production of gamma-linolenic acid by δδ-desaturase is described in USPN 5,552,306. The production of 8,11-ososadienoic acid using Mortierella alpina is described in USPN 5,376,541. The production of docosahexaenoic acid by dinoflagelate is described in USPN 5,407,957. The cloning of δδ-palmitoylacyl protein desaturase is described in PCT publication WO 96/13591 and USPN 5,614,400. Cloning of Δ6 desaturase from the borage is described in PCT publication WO 96/21022. The cloning of 99-desaturases is described in PCT Patent Application Publication No. WO 91/13972, EP 0 550 162 A1, EP 0 561 569 A2, EP 0 644 263 A2 and EP 0 736 598 A1 and USPN 5,057,419. Cloning of Δ12 desaturases from various organisms is described in PCT publication WO 94/11516 and USPN 5,443,974. The cloning of Δ15 desaturases from various organisms is described in PCT publication WO 93/11245.

SUMMARY OF THE INVENTION

Novel compositions and methods suitable for preparing long-chain polyunsaturated fatty acids or PUFAs are described. The compositions include nucleic acids encoding Δ6- and Δ12desaturase and / or polypeptides having Δ6- and Δ12desaturase activity, polypeptides and probes suitable for isolation and detection. The methods include culturing a host oragnism or constructing an elimination of concentrations in animal biosynthesis that express the introduced gene or genes encoding at least one desaturase, particularly zvláště6-, Δ9-, Δ12- or Δ15desaturase. The methods also include the use of nonsensical or gene disruptions to reduce or potentiate the expression of the desaturases desired. Regulation of expression of desaturase polypeptide (s) provides a relative increase in desaturated PUFAs of interest, as a result of altered enzymes and substrates that are included in PUFAs. The invention finds use, for example, in large-scale production of GLA, DGLA, ARA, EPA, DHA and SDA.

In a preferred embodiment of the invention there is provided a nucleic acid sequence comprising the nucleotide sequence shown in Figure 3A to E (SEQ ID NO: 1) or Figure 5A to D (SEQ ID NO: 3), a polypeptide encoded by the nucleotide sequence of Figure 3A. 3A to E (SEQ ID NO: 1) or Figure 5A to D (SEQ ID NO: 3) and a purified and isolated polypeptide comprising the amino acid sequence shown in Figure 3A to E (SEQ ID NO: 2) or 5A-D (SEQ ID NO: 4). In another embodiment, the invention provides an isolated nucleic acid that encodes a polypeptide having the amino acid sequence shown in Figure 3A to E (SEQ ID NO: 2) or Figure 5Ά to

D (SEQ ID NO: 4);

The invention provides an isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide that desaturates a fatty acid molecule at the sixth or twelfth carbon position from the carboxyl terminus, wherein said nucleotide sequence comprises an average A / T content of less than about 60%. In a preferred embodiment, the isolated nucleic acid is obtained from fungi such as Mortierella. More preferred are fungi of the genus Mortierella alpina.

In a preferred embodiment of the invention there is provided an isolated nucleic acid comprising the nucleotide sequence shown in Figure 3A to E (SEQ ID NO: 1) or Figure 5A to D (SEQ ID NO: 3). The invention further includes an isolated and purified polypeptide that desaturates a fatty acid molecule at the sixth or twelfth carbon position from the carboxyl terminus, wherein the polypeptide is a eukaryotic polypeptide or is derived from a eukaryotic polypeptide, wherein the preferred eukaryotic polypeptide is derived from fungi.

In another embodiment of the invention there is provided an isolated nucleic acid wherein the nucleotide sequence is set forth in Figure 3A to E (SEQ ID NO: 1) or Figure 5A to D (SEQ ID NO: 3). The invention further provides a purified and isolated polypeptide that desaturates a fatty acid molecule at the sixth or twelfth carbon position from the carboxyl terminus, wherein the polypeptide is or is derived from a eukaryotic polypeptide, wherein the preferred eukaryotic polypeptide is derived from fungi.

The invention further encompasses a nucleic acid that hybridizes to the nucleotide sequence set forth in Figure 3A to 3E (SEQ ID NO: 1) or Figure 5A to D (SEQ ID NO: 3). Preferably, an isolated nucleic acid having a nucleotide sequence having at least about 50% identity to the sequence of Figure 3A to E (SEQ ID NO: 1) or 5A to D (SEQ ID NO: 3). The invention further encompasses an isolated nucleic acid having a nucleotide sequence that exhibits at least about 50% homology to the sequence of Figure 5A to D (SEQ ID NO: 1) or Figure 5A to D (SEQ ID NO: 3). In a preferred embodiment, the nucleic acid of the invention comprises a nucleotide sequence that encodes the amino acid sequence shown in Figure 3A to D (SEQ ID NO: 2), selected from the group consisting of amino acid residues 50-53, 39-43, 172-176 , 204-213 and 390-402.

The invention further encompasses the construction of a nucleic acid comprising the nucleotide sequence indicated in Figure 3A to E (SEQ ID NO: 1) or Figure 5A to D (SEQ ID NO: 3) associated with a heterogeneous nucleic acid. In another embodiment, the invention provides a nucleic acid construct comprising

the nucleotide sequence shown in Figure 3A to E (SEQ ID NO: 1) or Figure 5A to D (SEQ ID NO: 3) operably linked to an expression control sequence that is functional in the host cell. The host cell is either a eukaryotic or a prokaryotic. Preferred eukaryotic host cells are those selected from the group consisting of a mammalian cell, an insect cell, a fungal cell, and an algal cell. Preferred mammalian cells include a bird cell, a preferred fungal cell is yeast, and a preferred algae cell is a seaweed cell. Preferred prokaryotic cells include those selected from the group consisting of bacteria, cyanobacteria, cells that contain bacteriophage and / or virus. Preferably, the recombinant host cell DNA sequence comprises a promoter that is functional in the host cell. Said promoter is inducible. In a more preferred embodiment of the invention, the microbial cell is a fungal cell of the genus Mortierella, more preferably a fungus sp. Mortierella alpina.

In another embodiment, the invention provides a nucleic acid construct that comprises a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence shown in Figure 3A to E (SEQ ID NO: 2) or Figure 5A to D (SEQ ID NO: 4). ), wherein the nucleotide sequence is operably linked to an expression control sequence that functions in a microbial cell, wherein the nucleotide sequence encodes a functional active polypeptide that desaturates the fatty acid molecule at a sixth or twelfth carbon position from the carboxyl terminus of the fatty acid molecule. The invention further provides a nucleic acid construct comprising a nucleotide sequence that encodes a functionally active β-desaturase, wherein the desaturase comprises an amino acid sequence that corresponds to or is complementary to all or part of the amino acid sequence shown in Figure 3A to E (SEQ ID NO: 2). ), wherein the nucleotide sequence is operably linked to a transcriptional control sequence functional in the host cell.

• ο • · ♦

The invention further encompasses the construction of a nucleic acid comprising the nucleotide sequence indicated in Figure 3A to E (SEQ ID NO: 1) or Figure 5A to D (SEQ ID NO: 3) associated with a heterogeneous nucleic acid. In another embodiment, the invention provides a nucleic acid construct comprising the nucleotide sequence shown in Figure 3A to E (SEQ ID NO: 1) or Figure 5A to D (SEQ ID NO: 3) operably linked to an expression control sequence that: is functional in the host cell. The host cell is either a eukaryotic or a prokaryotic. Preferred eukaryotic host cells are those selected from the group consisting of a mammalian cell, an insect cell, a fungal cell, and an algal cell. Preferred mammalian cells include a bird cell, a preferred fungal cell is yeast, and a preferred algae cell is a seaweed cell. Preferred prokaryotic cells include those selected from the group consisting of bacteria, cyanobacteria, cells that contain bacteriophage and / or virus. Preferably, the recombinant host cell DNA sequence comprises a promoter that is functional in the host cell. Said promoter is inducible. In a more preferred embodiment of the invention, the microbial cell is a fungal cell of the genus Mortierella, more preferably a fungus sp. Mortierella alpina.

In another embodiment, the invention provides a nucleic acid construct that comprises a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence shown in Figure 3A to E (SEQ ID NO: 2) or Figure 5A to D (SEQ ID NO: 4). ), wherein the nucleotide sequence is operably linked to an expression control sequence that functions in a microbial cell, wherein the nucleotide sequence encodes a functional active polypeptide that desaturates the fatty acid molecule at the sixth or twelfth carbon position from the carboxyl terminus of the fatty acid molecule. The invention further provides a nucleic acid construct comprising a nucleotide sequence that encodes a functionally active β-desaturase, wherein the desaturase comprises an amino acid sequence that corresponds to * or is complementary to all or part of the amino acid sequence shown in Figure 3A to E (SEQ ID NO. 2), wherein the nucleotide sequence is operably linked to a transcriptional control sequence functional in a host cell.

The invention further encompasses the construction of a nucleic acid comprising the nucleotide sequence indicated in Figure 3A to E (SEQ ID NO: 1) or Figure 5A to D (SEQ ID NO: 3) associated with a heterogeneous nucleic acid. In another embodiment, the invention provides a nucleic acid construct comprising the nucleotide sequence shown in Figure 3A to E (SEQ ID NO: 1) or Figure 5A to D (SEQ ID NO: 3) operably linked to an expression control sequence that: is functional in the host cell. The host cell is either a eukaryotic or a prokaryotic. Preferred eukaryotic host cells are those selected from the group consisting of a mammalian cell, an insect cell, a fungal cell, and an algal cell. Preferred mammalian cells include a bird cell, a preferred fungal cell is yeast, and a preferred algae cell is a seaweed cell. Preferred prokaryotic cells include those selected from the group consisting of bacteria, cyanobacteria, cells that contain bacteriophage and / or virus. Preferably, the recombinant host cell DNA sequence comprises a promoter that is functional in the host cell. Said promoter is inducible. In a more preferred embodiment of the invention, the microbial cell is a fungal cell of the genus Mortierella, more preferably a fungus sp. Mortierella alpina.

In another embodiment, the invention provides a nucleic acid construct that comprises a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence shown in Figure 3A to E (SEQ ID NO: 2) or Figure 5A to D (SEQ ID NO: 4). ), wherein the nucleotide sequence is operably linked to an expression control sequence that functions in a microbial cell, wherein the nucleotide sequence encodes a functional active polypeptide that desaturates the fatty acid molecule at the sixth or twelfth carbon position from the carboxyl terminus of the fatty acid molecule. The invention further provides a nucleic acid construct comprising a nucleotide sequence that encodes a functionally active 66-desaturase, wherein the desaturase comprises an amino acid sequence that matches or is complementary to all or part of the amino acid sequence shown in Figure 3A to E (SEQ ID NO: 2). ), wherein the nucleotide sequence is operably linked to a transcriptional control sequence functional in the host cell.

The invention further encompasses the construction of a nucleic acid comprising a nucleotide sequence that encodes a functional, active A12-desaturase having an amino acid sequence that matches or is complementary to all or part of the amino acid sequence shown in Figure 5A to D (SEQ ID NO: 4). ), wherein the nucleotide sequence is operably linked to a transcriptional control sequence functional in the host cell. The host cell is either a eukaryotic or a prokaryotic. Preferred eukaryotic host cells are those selected from the group consisting of a mammalian cell, an insect cell, a fungal cell, and an algal cell. Preferred mammalian cells include a bird cell, a preferred fungal cell is yeast, and a preferred algae cell is a seaweed cell. Preferred prokaryotic cells include those selected from the group consisting of bacteria, cyanobacteria, cells that contain bacteriophage and / or virus. Preferably, the recombinant host cell DNA sequence comprises a promoter that is functional in the host cell. Said promoter is inducible. In a more preferred embodiment of the invention, the microbial cell is a yeast cell such as a Saccharomyces cell.

In a preferred embodiment of the invention there is provided a recombinant host cell comprising at least one copy of a nucleic acid that encodes a functionally active Mortierella alpine fatty acid desaturase having the amino acid sequence shown in Figure 3A to E (SEQ ID NO: 2), wherein the cell or its parent cell is transformed with a vector comprising said DNA sequence and wherein the DNA sequence is operably linked to an expression control sequence.

In a preferred embodiment of the invention, the cell is a microbial cell enriched in 18: 2 fatty acids, especially a microbial cell from a genus selected from the group consisting of prokaryotic and eukaryotic cells. In another embodiment of the invention, the microbial cell of the invention comprises an expression control sequence that is endogenous to the microbial cell.

The invention also encompasses a method of producing GLA in a microbial cell culture, the method comprising culturing host cells, the culture comprising a plurality of cells comprising one or more nucleic acids encoding a polypeptide that converts LA to GLA, wherein the one or more nucleic acids is operatively linked to an expression control sequence under conditions wherein said one acid is expressed, wherein GLA

In several other embodiments of the invention, a polypeptide that is a functionally active enzyme that desaturates a fatty acid molecule at the sixth carbon position from the carboxyl terminus of the fatty acid molecule is used; one or more nucleic acids are obtained from Mortierelle alpina and the substrate of said polypeptides is delivered exogenously. Host cells are microbial cells. The microbial cells are yeast cells such as saccharomyces cells. The culture conditions are inducible.

The invention also includes an oil comprising one or more PUFAs. The amount of said one or more PUFAs is about 0.3 to 30% arachidonic acid (ARA), about 0.2 to 30% dihomo-γ-linolenic acid (DGLA), and about 0.2 to 30% γ-linolenic acid (GLA) ). A preferred oil of the invention is one wherein the ratio of ARA: DGLA: GLA is about 1.0: 19.0: 30 to 6.0: 1.0: 0.2. Another preferred embodiment of the invention is or more nucleic acids in the host cell.

to produce a preferred pharmaceutical composition comprising oils in a pharmaceutically acceptable carrier. Further described is a nutritional composition comprising the oils of the invention. The nutritional compositions of the invention are preferably administered parenterally or internally to a mammalian host. The preferred composition of the invention is infant formula. In a preferred embodiment of the invention, the infant formula is in solution or solid form. This nutrition occurs as a dietary supplement and the oil is in the form of capsules. The oil of the invention need not contain any particular components of other oils obtained by microbial cells, such as a yeast cell.

The invention further includes a process for desaturating a fatty acid. A preferred embodiment of the method comprises culturing a recombinant microbial cell of the invention under conditions suitable for expression of the polypeptides encoded by said nucleic acid, wherein the host cell further comprises a fatty acid substrate suitable for the polypeptide. In a preferred embodiment of the invention, the fatty acids desaturated by the methods of the invention and comprise an oil containing the fatty acid produced according to the invention.

The invention further encompasses a peptide sequence which is a purified nucleotide substantially related to or homologous to the nucleotide peptide sequence present in SEQ ID NOS: 1-40. The invention further provides methods of using the sequences present in SEQ ID NOS: 1-40 as a probe to identify related sequences, as components of expression systems and as components of systems useful for producing transgenic oil.

The invention further provides nutritional, dietary supplements or food substitutes in solution or solid form containing the long chain fatty acids of the invention. These nutrients may be administered to a human or animal.

The nutritional or supplements of the invention may further comprise at least one macronutrient selected from the group consisting of mono- and lactose oil, coconut milk electrodialysed, soybean oil, canola oil, diglycerides, glucose, low-fat, low-fat, low-fat, whey, soy protein and other protein hydrolysates.

The nutrients of the invention may further comprise at least one vitamin selected from the group consisting of a vitamin Ά, C, D, E and B complex and at least one mineral selected from the group comprising calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium and iron.

The invention further provides a method of treating a patient suffering from insufficient intake or production of polyunsaturated fatty acids comprising administering to the patient of the invention a food substitute in an amount sufficient to treat the patient.

The invention further provides cosmetic and pharmaceutical compositions of the materials of the invention.

The invention further encompasses transgenic oils on pharmaceutically acceptable carriers. The invention further provides nutritional supplements, cosmetic agents and infant formulas containing transgenic oils.

The invention further encompasses a method of obtaining altered long chain polyunsaturated fatty acid biosynthesis comprising: culturing a microbe having cells containing a transgene that encodes a product expressed by the transgene. This product desaturates the fatty acid molecule at a carbon 6 or 12 position from the carboxyl terminus of the fatty acid molecule, where the transgene is operably linked to an expression control sequence, altering the biosynthesis of long-chain polyunsaturated fatty acids in cells.

The invention further provides pharmaceutical compositions comprising at least one nutrient selected from the group consisting of a vitamin, a mineral, a carbohydrate, a sugar, an amino acid, a free fatty acid, a phospholipid, an antioxidant, and a phenolic compound.

Definition :

A5-desaturase: A5-desaturase is an enzyme that introduces a double bond between the fifth and sixth carbon from the carboxyl terminus of a fatty acid molecule.

Δδ-desaturase: Δ6 desaturase is an enzyme that introduces a double bond between the sixth and seventh carbon from the carboxyl terminus of a fatty acid molecule.

99-desaturase: Δ9 desaturase is an enzyme that introduces a double bond between the ninth and tenth carbon from the carboxyl terminus of a fatty acid molecule.

A12-desaturase: Δ12 desaturase is an enzyme that introduces a double bond between 12 and 13 carbon from the carboxyl terminus of a fatty acid molecule.

Fatty acids: Fatty acids are a class of substances containing a long hydrocarbon chain and a terminal carboxyl group. Fatty acids include:

Fatty acids 12: 0 Kys. laurová 16: 0 Kys. palmitová 16: 1 Kys. palmitolejová 18: 0 Kys. stearová 18: 1 Kys. oil 9-18: 1 18: 2 ,95.9 Kys. taxolejová 5: 9-18: 2 18: 2 ,96.9 Kys. 6,9- octadecadiene 6: 9-18: 2 18: 2 Kys. linoleum 9: 12-18: 2 (LA) 18: 3-6, 9, 12 Kys. gamma-linoleum Rev. 6, 9, 12-18: 3 (GLA) 18: 3 ,95,9,12 Kys. pinolenová 5,9,12-18: 3 18: 3 Kys. alpha-linolene Δ9, 12.15-18: 3 (ALA) 18: 4 Kys. stearidone ,96,9,12,15-18: 4

• ·

(SDA) 20: 0 Kys. arachidone 20: 1 Kys. ikoscenová 22: 0 Kys. behehová 22: 1 Kys. eruková 22: 2 Kys. dokasadienová 20: 4 ω6 Kys. arachidone ,85.8,11.14-20: 4 (ARA) 20: 3 ω6 ω6-icosatriene dihomo-gama- linolenová Δ8,11,14-20: 3 (DGLA) 20: 5 ω5 Ikosapentanová (timnodon) Δ5,8,11,14,17- 20: 5 20: 3 ω3 o> 3-icosatriene 11,16,17-20: 3 20: 4 ω3 co3-icosatetraene ,107,10,13,16,19-22: 5 (cú3DPA) 22: 5 ω3 Dokosapentanová 7, 10,13,16,19-22: 5 (o3DPA) 22: 6 ω3 Dokasahexanová (cervonic acid) Δ4,7,10,13,16,19- 22: 6 24: 0 Lignocerová kys.

In view of these definitions, the invention describes novel DNA sequences, DNA constructs, methods, and compositions that allow modification of long-chain polyunsaturated fatty acids in, for example, microbial cells or animals. Host cells are manipulated to express the sense and antisense transcript of the DNA that encodes the polypeptide (s) that catalyze the desaturation of the fatty acid. The substrate (s) of the expressed enzymes may be produced in the host cell or may be delivered exogenously. In order to achieve expression, the transformed DNA is operably linked to the transcription and translation start site and the termination regulatory regions that are functional in the host cell. Constructs containing the expressed gene may be incorporated into the genome of the host cell or may replicate separately in the host cell. In the case of linolenic acid (LA) production, commonly used expression cassettes include a cassette that provides 1212-desaturase activity, particularly in a host cell that produces or can absorb oleic acid (US Patent No. 5,443,974). LA production can also be increased by preparing an expression cassette for 99-desaturase, where enzymatic activity is limiting. In the case of ALA production, commonly used expression cassettes include a cassette that exhibits Δ15 or β-desaturase activity, particularly in a host cell that produces or can absorb LA. In the case of GLA or SDA production, the expression cassettes commonly used include a cassette that exhibits δδ-desaturase activity, particularly in a host cell that produces or may engulf LA or ALA.

The production of a polyunsaturated fatty acid type 6, such as LA or GLA, is preferred in a host microorganism or animal that is unable to produce ALA.

A host suitable for ALA production can be removed, reduced or obtained by inhibiting des15 or ω3-type desaturase activity (Figure 2). This can be achieved by standard selection.

Endogenous desaturase activity may be affected by preparing an expression cassette to prepare an antisense Δ15 or ©3 transcript expression cassette, disrupting the Δ15- or ©3desaturase target gene through insertion, substitution, deletion of all or part of the target gene, or by adding Δ15- or 33-desaturase inhibitor. Similarly, LA or ALA production is favored in a microorganism or animal that exhibits Δ6 desaturase activities by providing an expression cassette of a nonsense Δ6 transcript by disrupting the Δδ-desaturase gene or by using a β-desaturase inhibitor.

• · ·

Microbial production of fatty acids

Microbial production of fatty acids has several advantages over isolation from natural sources such as fish or plants. Many microbes are known to contain highly simplified oil compositions as compared to compositions containing higher organisms, thereby simplifying the purification of the desired components. Microbial production is not subject to fluctuations caused by external variables such as weather and food supply. The microbially produced oil is substantially free of contamination of environmental pollutants. In addition, microbes provide PUFAs in certain forms that may have specific uses. For example, spirulina may provide PUFAs predominantly in the first and third positions of triglycerides. Preferably, fatty acids are liberated from these positions by pancreatic lipase digestion. Upon ingestion of Spirulina-derived triglycerides in humans and animals, these PUFAs are released by pancreatic lipases, as free fatty acids, and are thus directly available, for example, to the development of the infant's brain. In addition, microbial oil production can be manipulated by controlling the culture conditions, particularly by preparing certain substrates for microbially expressed enzymes or by adding substances that suppress the desired biosynthesis pathways. In addition to these advantages, the production of fatty acids from recombinant microbes provides the ability to alter the naturally occurring profile of microbial fatty acids by providing a new synthetic pathway in the host or suppressing unwanted pathways, increasing the amount of PUFA or conjugated forms thereof and suppressing undesirable PUFA.

Fatty acid production in animals

Animal fatty acid production also has several advantages.

Expression of desaturase genes in animals can produce considerably increased levels of PUFAs in animal tissue, which they do

Their isolation from these tissues is economical. For example, when PUFAs are expressed in animal milk, methods of isolating PUFAs from milk are well established. In addition to the possibility of obtaining a source for purification of the desired PUFAs, animal milk may be manipulated by expressing desaturase genes, either alone or in combination with other human genes, to produce animal milk containing PUFAs that substantially corresponds to human milk during various stages of infant development. Animal milk adapted for human ingestion may serve as infant food in cases where breastfeeding is not possible or breastfeeding is not desirable or in cases of malnutrition or disease.

Depending on the host cell, the availability of the substrate and the desired end product (s), several polypeptides, particularly desaturases, are of great interest. The term "desaturase" refers to a polypeptide that can desaturate one or more fatty acids to produce a mono- or polyunsaturated fatty acid or precursor thereof. Of particular interest are polypeptides that can catalyze the conversion of stearic acid to oleic acid, oleic acid to LA, LA to ALA, LA to GLA, or ALA to SDA. They are enzymes that desaturate at positions Δ9, Δ12, (ω6), Δ15, (ω3) or Δ6. The term "polypeptide" means any chain of amino acids, regardless of length or post-translational modifications, such as glycosylation or phosphorylation. When selecting a specific polypeptide having desaturation activity, it is necessary to consider the optimum pH of the polypeptide, whether the polypeptide is a speed-limiting enzyme or a component thereof, whether the desaturase used is essential for the synthesis of the desired fatty acids and / or whether the polypeptides require co-factors. The expressed polypeptide preferably has parameters compatible with the biochemical environment of its location in the host cell. For example, a polypeptide may compete for substrate with other enzymes in a host cell. The K _m and specific activity of the polypeptide are analyzed to determine whether these values are a determining criterion for the suitability of a given polypeptide in modifying PUFA production in a given host cell. The polypeptide used in a particular situation is one that is functional under the conditions in the host cell, but otherwise it may be a polypeptide having desaturase activity that is capable of modifying the relative production of the desired PUFAs.

In the case of linolenic acid production, the DNA sequence encodes a polypeptide that exhibits 1212-desaturase activity.

In cases of GLA production from linolenic acid, said DNA sequence encodes a polypeptide having A66-desaturase activity.

In certain instances, expression of A66-desaturase activity may be associated with expression of 1212-desaturase activity and 1515-desaturase activity may be removed from the host cell by, for example, preparing a transcriptional cassette for producing antisense sequences for the 1515-desaturase transcription product by damaging the gene A15 desaturase or by using a host cell that naturally exhibits low mutation A15 desaturase activity. Inhibition of desired desaturation pathways can also be achieved using specific desaturase inhibitors as described in US Patent 4,778,630. Also, the host cell for the expression of A12-desaturase may exhibit high A12-desaturase activity or may be mutated for this purpose. The choice of combination of cartridges used may depend in part on the PUFA profile and / or the desaturase profile in the host cell. When the host cell expresses 1212-desaturase activity and lacks or eliminates 1515-desaturase activity, overexpression of A66-desaturase alone is generally sufficient to produce increased GLA production. If the activity of 99-desaturase, then desaturase and A66-desaturase can produce increased GLA production.

the host cell expresses A12-desaturase A1220 expression

If the cell does not exhibit or is limiting A9-desaturase activity, an 99-desaturase expression cassette may be used. FIG. 2 shows a schematic representation of the synthesis of arachidonic acid (20: 4 Δ ^{5 '8' 11 '14)} _{of PY]} _p _n y stearic (18: 0). The key enzyme in this pathway is β-desaturase, which converts linolenic acid to γ-linolenic acid. Furthermore, the conversion of α-linolenic acid (ALA) to stearidonic acid by β-desaturase is shown.

Sources of polypeptides exhibiting desaturation activity

Sources of polypeptides exhibiting desaturation activity and oligonucleotides encoding such polypeptides are organisms that produce the desired polyunsaturated fatty acid. Examples of microorganisms having the ability to produce GLA or ARA can be used as sources of Δ6- or Δ12desaturase activity. Such microorganisms include, for example, those belonging to the genus Mortierella, Conidiobolus, Pythium, Phytophathora, Penicillium, Porphyridium, Coidosporium, Mucor, Fusarium, Aspergillus, Rhodotorula, and Entomophthora. In the genus Porphyridium is interesting Porphyridium cruentum. Mortierella elongata, Mortierella exigua, Mortierella hygrophila, Mortierella ramanniana, var. angulispora and Mortierella alpina. In the genus Mucor Mucor circinelloídes and Mucor j avanicus are of particular interest.

DNAs encoding desaturases of interest can be identified in various ways. The source of the desired desaturase, for example a genomic DNA library or Mortierella cDNA library, can be tested with detectable enzymatically or chemically synthesized probes, which can be prepared from non-naturally occurring DNA, RNA or nucleotides, or a mixture thereof. The probes can be synthesized enzymatically from known DNA desaturases and are suitable for hybridization methods of normal or reduced stringency. Oligonucleotide probes can also be used as sources of testing and can be based on known desaturase sequences, including sequences that are conserved among known desaturases, or on peptide sequences obtained from the desired purified antibody.

Protein-based oligonucleotide probes. The amino acid sequences on the sequences may degenerate to emphasize the degeneracy of the genetic code or may be rearranged in favor of preferred codons of the source organism. Oligonucleotides can be used as primers for PCR from reverse transcribed rnRNA from a known or dubious source. The PCR product may be full-length cDNA or may be used to generate a probe that serves to obtain full-length cDNA. Alternatively, the desired protein can be completely sequenced and total DNA synthesis encoding said polypeptide is performed.

If necessary, genomic DNA or cDNA is isolated, which can be sequenced by known methods. Such sequencing methods are known to be flawed. Therefore, multiple sequence sequencing has become routine, and it is still necessary to count on a measurable error rate in the resulting deduced sequence, especially in regions that exhibit repeats, extensive secondary structure, or unusual composition of bases, such as regions with high GC content. If discrepancies occur, re-sequencing can be performed and special methods can be used. Said special methods may include altering the sequencing conditions using: different temperatures, different enzymes, proteins that alter the ability of oligonucleotides to form higher order structures, altered nucleotides such as ITP or methylated dGTP, different gel compositions such as adding formamide, different primers or localized primers at different distances from the problematic region or different templates such as single-stranded DNA. Sequencing of rnRNA can also be used.

4 4 4 45 «» »·

In most cases, some or all of the polypeptide coding sequences showing desaturation activity originate from a natural source. In some situations, it is necessary to modify all or part of the codons, for example, to enhance expression using host-preferred codons. Hosts can be determined from the codons that are most commonly found in proteins that are expressed in the largest amount in a particular host species. The coding sequence of a polypeptide exhibiting desaturation activity may be synthesized in whole or in part. All or part of the DNA can also be synthesized to remove any destabilizing sequences or regions of secondary structure that will occur in the transcribed mRNA. All or parts of the DNA can also be synthesized to change the composition to those preferred by the host cell of interest. Methods for synthesizing sequences and linking sequences are reported in the literature. In vitro mutagenesis and selection, or site-directed mutagenesis may then be used to obtain mutations of naturally occurring desaturase genes to produce a polypeptide that exhibits desaturation activity in vivo with physical and kinetic parameters more suited to host cell function. longer half-life

Cell by other methods, desired polyunsaturated fatty acids higher production rate.

Desaturase of the organism Mortierella alpina

Of interest is Mortierella alpina Δδ-desaturase, which has 457 amino acids and has a predicted molecular weight of 51,800. The amino acid sequence is shown in Figure 3. The gene encoding Mortierella alpina A6-desaturase can be expressed in transgenic microorganisms or in animals so that a stronger synthesis of GLA from linolenic acid and staearidonic acid from ALA is achieved. Other DNAs that are substantially identical to Mortierella alpina β-desaturase DNA or that encode polypeptides that are substantially identical to Mortierella alpina β-desaturase polypeptide may also be used. The term "substantially identical" means an amino acid sequence or a nucleic acid sequence having, for increased preference, at least 60%, 80%, 90% or 95% homology with the Mortierella alpina β-desaturase amino acid sequence or with a nucleic acid sequence encoding an amino acid sequence. In the case of polypeptides, the length of the sequences to be compared is generally at least 16 amino acids, preferably at least 20 amino acids, more preferably 35 amino acids. In the case of nucleic acids, the length of the sequences to be compared is generally at least 50 nucleotides, preferably at least 60 nucleotides and more preferably at least 75 nucleotides, and most preferably 110 nucleotides. Typically, homology is measured using sequence analysis software such as Sequence Analysis software from Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705, MEGAlign (DNAStar, Inc., 1228 S. Park) St., Madison, Wisconsin 53715) and MacVector (Oxford Molecular Group, 2105 S. Bascom Avenue, Suite 200, Campbell, California 95008). Such software pairs similar sequences, thereby assigning different degrees of homology to different substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions in the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid, glutamic acid, asparagine, and glutamine; serine and threonine; lysine and arginine; phenylalanine and tyrosine. Substitutions may also be made based on conservative hydrophobicity or hydrophility (Kyte and Doolittle, J. Mol. Biol. 157: 105-132, 1982) or on the ability to predict a similar secondary structure of a polypeptide (Chou and Fasman, Adv. Enzymol. 47: 45-148, 1978).

• ·

Other desaturases

The invention describes related desaturases derived from the same or another organism. Such related desaturases include variants of the described Δ6- or 1212-desaturase occurring in the same or different Mortierella species as well as homologues of the Δ6- or 1212-desaturase from other species. The invention further provides desaturases which, although not substantially identical to the β- or 1212-desaturase of Mortierella alpina, desaturate the fatty acid molecule at the 6 and 12 carbon positions from the carboxyl terminus of the fatty acid molecule. Related desaturases can be identified by their ability to function in the same way as the described desaturases, which are still capable of effectively converting LA to GLA, ALA to SDA, and oleic acid to LA. Related desaturases can also be identified by searching the sequence database. By hybridizing with a probe derived from the described desaturase with a library constructed from the source organism or by RT-PCR from mRNA from the source organism and primers based on the described desaturase, sequences homologous to the described desaturase are sought. Such desaturases include human desaturases, Dictyostelium discoideum, and Phaeodactylum tricornum.

Desaturase polypeptide regions that are important for desaturation activity can be determined by routine mutagenesis, expression of the resulting mutant polypeptides, and determination of their activities. Mutants may include deletions, insertions, and point mutations or combinations thereof. Typical functional analysis begins with deletion mutagenesis to determine the N- and C-terminal protein constraints necessary for protein function. Internal deletions, insertions, or point mutations are then performed to determine the regions necessary for the functions of said protein. Further, other methods such as cassette mutagenesis or total synthesis may be used. Deletion mutagenesis is performed, for example, using exonucleases, whereby the coding regions of the 5 'and 3' ends are sequentially removed. Kits are available for performing such procedures. After the deletion, the coding region is complemented by ligation of oligonucleotides containing the start and stop codons. Alternatively, oligonucleotides encoding the start and end codons are incorporated into the coding region using various methods including site-directed mutagenesis, mutagenic PCR or ligation to DNA that is cleaved at existing restriction sites. Similarly, internal deletions can be generated by a variety of methods, including the use of existing restriction sites in DNA, the use of mutagenic primers by site-directed mutagenesis or mutagenic PCR, or by ligation to DNA cleaved at existing restriction sites. Similarly, internal deletions can be performed using various methods involving the use of existing restriction sites in DNA using mutagenic primers via site-directed mutagenesis or mutagenic PCR. Incorporation is performed using methods such as linker searched mutagenesis, site directed mutagenesis, or mutagenic PCR. Point mutations are performed by methods such as site directed mutagenesis or mutagenic PCR.

Chemical mutagenesis can also be used to identify regions of desaturase polypeptides that are important for activity. The mutated construct is expressed and tested for the ability of the altered protein to function as a desaturase. Such structure-function analysis can determine which regions can be deleted, in which regions insertion is tolerated, and which point mutations give rise to a mutant protein that functions in much the same way as natural desaturase. The invention describes all such mutant proteins and nucleotide sequences encoding these proteins.

Expression of desurase genes

Once the DNA encoding the desaturase polypeptide has been obtained, it is placed in a vector capable of replicating in the host cell or propagated in vitro by a method such as PCR or long PCR. Replicating vectors may include plasmids, phages, viruses, cosmids, and the like. Desired vectors include vectors useful in gene mutagenesis or gene expression in host cells. The long PCR method performs in vitro propagation of a large construct by altering the gene to mutagenize or add expression signals. Thus, the resulting constructs may be propagated in vitro without using a replication vector or a host cell.

In order to express desaturase polypeptides, functional transcriptional and translational initiation and termination regions are operably linked to DNA encoding the desaturase polypeptide. Expression of the polypeptide coding region can take place in vitro or in a host cell. The transcription and translation initiation and termination regions have been derived from various sources, including expressed DNA, genes known or expected to be expressed in the desired system, expression vectors, chemical synthesis, or endogenous locus in the host cell.

Expression in vitro

For example, in vitro expression can be accomplished by placing the desaturase polypeptide coding region in an expression vector designed for in vitro use and adding rabbit lysate of reticulocytes and cofactors. If desired, labeled amino acids may also be included. Such in vitro expression vectors may provide some or all of the expression signals necessary for the system employed. These methods are well known in the art and system components are commercially available. In the reaction mixture, the presence of polypeptides can be directly tested, for example, by determining its activity, or the synthesized polypeptide can be purified and then tested.

Expression in the host cell • ·

Expression in the host cell may be temporary or stable. Temporary expression occurs by introducing constructs that contain expression signals functional in the host cell, but said constructs do not replicate and sparsely integrate in the host cell, or the host cell is scarce. Temporary expression can also be achieved by inducing the activity of a regulatable promoter that is operably linked to the gene. Such inducible systems often show poor expression. Stable expression can be achieved by introducing a construct that can be incorporated into the host genome or can self-replicate in the host cell. Stable gene expression can be selected using a selectable marker that is found in the expression construct or is transfected together with the expression construct. This is followed by selection of cells expressing the marker. Stable expression results from the incorporation of the construct. Integration of constructs can occur randomly in the host genome or can be targeted through the use of constructs containing regions of homology to the host genome, which is sufficient for recombination with the host locus. If the constructs are targeted to an endogenous locus, then the endogenous locus may provide all or some of the transcriptional or translational regulatory regions.

If it is necessary to enhance expression of the desaturase polypeptide in the source organism, several methods may be used. Additional genes encoding a desaturase polypeptide can be introduced into the host organism. Expression from the natural desaturase locus may also be enhanced by homologous recombination, for example by incorporating a stronger promoter into the host genome, removing destabilizing sequences from either the mRNA or the encoded protein by deleting said information from the host genome or adding stabilizing sequences to the mRNA (USPN 4,910,141).

If it is necessary to express more than one different gene then the introduced genes can be propagated in the host cell beyond

The use of replication vectors or by incorporation into the host genome. If two or more genes are expressed from a separate replication vector, each vector must have a different replication pattern. Each established construct, whether incorporated or not, should have a different mode of selection and should not be homologous to other constructs, as a condition of stable expression, thus avoiding rearrangement of elements between constructs. The correct choice of regulatory regions, the methods of selection and the method of propagation of the introduced construct can be determined experimentally such that all introduced genes can be expressed in the strength required to synthesize the desired products.

When the host cell is a yeast, it also provides transcriptional and translational regions. Transcriptional initiation regulatory regions may be derived from genes involved in the glycolytic pathway, such as alcohol dehydrogenase, glycerylaldehyde-3-phosphate dehydrogenase (GPD), phosphocucoisomerase, phosphoglycerate kinase, etc., or from controllable genes such as acid phosphatase, lactase, metallothionine etc. In a certain situation, depending on whether constitutive or induced transcription is required, depending on the promoter efficiency in conjunction with the open reading frame, the ability to associate a strong promoter with a control region from a different promoter that allows inducible transcription to simplicity of construction and the like, any of the regulatory sequences can be used. Of great interest is the promoters that are activated in the presence of galactose. Galactose-inducible promoters (GAL1, GAL7 and GAL10) can be used for strong and regulated protein expression in yeast (Lue et al., Mol. Cell. Biol. Vol. 7, P. 3446, 1987); Johnston, Microbiol. Roar. Vol. 51, p. 458 (1987). Transcription from GAL promoters is activated by the GAL4 protein, which binds to the promoter region and activates transcription when galactose is present.

<·

29 • * · »10 · · • · · • · · • «· «· · · · • · · · · · · · · · • «· · · • · · · ······ • · · In the absence of galactose anatagonista GAL80 se binds to GAL 4 a prevents GAL4 from being activated transcription. Addition galactose defends GAL80 inhibit GAL4 activation

The nucleotide sequences surrounding the ATG translation initiation codon have been found to cause expression in yeast cells. If necessary, the polypeptide is very poorly expressed in yeast. The nucleotide sequences of the exogenous genes may be modified to include an efficient yeast translation initiating sequence, thereby obtaining optimal gene expression. In the case of expression in Saccharomyces, this can be accomplished by site-directed mutagenesis of an inefficiently expressed gene by fusing into the endogenous Saccharomyces gene. A highly expressed gene, such as a lactase gene, is preferred.

The termination region can be obtained from the 3 ' region of the gene from which the initiation region was derived or from a different gene. A large number of termination regions are known which are useful in different hosts of the same or different genus and species. The terminating region is usually selected based on whether it is preferred or not, rather than based on a particular property. It is preferred that the termination region be obtained from a yeast gene, particularly from Saccharomyces, Schizosaccharomyces, Candida or Kluyveromyces. It is known that the 3 'regions of two mammalian genes, γ interferon and α.2 interferon, function in yeast.

Introduction of constructs into host cells

The gene-containing construct can be introduced into the host cell in a standard manner. These methods include transformation, transfection, transduction, introduction, electroporation, any other method, protoplast fusion, lipofection, conjugation, infection, ballistic microinjection, scraping, or by introducing a gene into a host cell. Transformation methods used include lithium acetate transformation (Methods in Enzymology, Vol. 194, P. 186-187, 1991). It is preferred that the manipulated host cell that absorbs the DNA sequence or said construct be termed "transformed or" recombinant.

The host will contain at least one copy of the expression construct. However, it may contain two copies or more, depending on whether the gene is incorporated into the genome, amplified or is present on an extrachromosomal element that has multiple copies. If the host is yeast, then four basic types of yeast plasmid integrating plasmids (YIp), yeast (YRp), yeast centromeric plasmids vectors: yeast replication plasmids (YCp) and yeast episomal plasmids (YEp) can be used. YIp lacks the origin of yeast replication and must multiply as integrated elements in the yeast genome. YRβs have a self-replicating yeast replicating sequence derived from chromosomes and propagate with a median copy number (20-40) as self-replicating unstable segregating plasmids. YCps have an origin of replication and a centromeric sequence and propagate with a small copy number (10-20) as self-replicating stable segregation plasmids. YEp plasmids have an origin of replication from a 2 µm yeast plasmid and propagate with a high copy number as self-irregularly segregating plasmids. The presence of plasmids can be ascertained by selection based on labeling in the plasmid. Of interest are yeast vectors pYES2 (YEp plasmid available from Invitrogen, conferring uracil prototrophy and galactose-inducible GAL1 promoter), PRS425-pG1 (YEp plasmid available from Dr. THChang, Associate Professor of Molecular Genetics, Ohio State University, this plasmid contains the constitutive promoter GPD and confers prototrophy of leucine) and pYX424 (plasmid YEp has the constitutive promoter TP1 and confers prototrophy of leucine; Alber, T. and Kawasaki, G. (1982). J. Mol. and Appl. Genetics 1: 419) .

The transformed host cell can be identified by selecting a marker that is included in the established construct.

Alternatively, a separate marker construct may be introduced into the host cell together with the desired construct.

Typically, transformed hosts were selected based on their ability to grow on the selection medium. Selection media may contain an antibiotic and may lack the factor necessary for the growth of the untransformed host. Such a factor may be a nutrient or a growth factor. Such a introduced marker gene can confer antibiotic resistance or encode a substantial growth factor or enzyme and allow growth on selection medium when expressed in a transformed host. The selection of the transformed host can also occur when the expressed marker protein can be detected directly or indirectly. The marker protein may be expressed alone or as a fusion with another protein. The marker protein can be detected based on its enzymatic activity. β-galactosidase can convert the substrate X-gal into a colored product and luciferase can convert luciferin into a light-emitting product. The marker protein can be detected by producing light or changing characteristics. The green fluorescent protein of Nequorea victorie is irradiated with blue light. To detect or the molecular tag present fluoresces if the marker protein, for example, in the protein

Cells can be used with antibodies expressing a marker protein or tag selected based on visual observation or in a manner such as FACS or virgin using antibodies. Any marker that is functional in yeast may be used in the selection of yeast transformants. An interesting marker is resistance to kanamycin and aminoglycoside G418, as well as the ability to grow on media that does not contain uracil, leucine or tryptophan.

FA6- and Δ12-desaturase-mediated PUFA production can occur in either prokaryotic or eukaryotic host cells. Prokaryotic cells include Eschericia, Bacillus, Lactobacillus, cyanobacteria and the like. Eukaryotic cells include mammalian cells such as lactating animal cells, avian cells such as chicken cells and other cells responsible for genetic manipulation including insect, fungal and algae cells. The cells may be cultured or formed as part or all of a host organism including animals. Viruses and bacteriophages can also be used in conjunction with cells, particularly gene transfer, cell targeting and selection, to produce PUFAs. In a preferred embodiment of the invention, the host is any microorganism or animal that produces and / or assimilates exogenously administered substrate (s) for Δ6- and / or 1212-desaturase and preferably produces a large amount of one or more substrates. Examples of host animals include mice, rats, chickens, quails, turkeys, bovine, sheep, pigs, goats, etc., which are responsible for genetic manipulation and cloning to rapidly increase the transgenic expression population. In animals, the desaturase transgene (s) can be engineered by modifying the regulatory regions of the gene to be expressed in target organelles, tissues, and body fluids. There is great interest in PUFA production in the milk of the host animal.

Expression in yeast

Examples of host microorganisms include Saccharomyces cerevisiae, Saccharomyces carlsbergensis nbeo other yeasts such as Candida, Kluyveromyces or other fungi such as filamentous fungi such as Aspergillus, Neurospora, Penicillium etc. The desired characteristics of the host microorganism are as follows: it is well genetically characterized; strong expression of the product using ultra-high density fermentation and is on the GRAS list (generally safe), which means that the end product is suitable for human administration. Also of interest is the use of yeast as a host organism according to the invention, in particular baker's yeast (S. cerevisiae). Particularly interesting strains are SC334 (Mat apep4-3 prbl-1122 ura3-52 leu2-3, 112 regl-501 gall; Gene 83: 57-64, 1989, Hovland P. et al.), YTC34 (and ade2-101 his3A200 Llys2-801 ura3-52; obtained from Dr. THChang, Associate Professor of Molecular Genetics, Ohio State University), YTC41 (a / α ura352 / ura3 = 521ys2-80l / lys2-801 ade2-101 / ade2-101 trpl-ΔΙ / trpl-ΔΙ his3A200 / his3A2001eu2Al / leu2Al; obtained from Dr. THChang, Associate Professor of Molecular Genetics, Ohio State University), BJ1995 (obtained from Yeast Genetic Stock Center, 1021 Donner Laboratory, Berkley, CA 94720), INVSC1 ( Mat and hiw3A1 leu2 trpl-289 ura3-52, obtained from Invitrogen, 1600Faraday Ave., Carlsbad, CA 92008) and INVSC2 (Mat and his3A200ura3-167; obtained from Invitrogen).

Expression in specie birds

In the production of PUFAs in avian species and in avian cells such as chickens, turkeys, quails and ducks, gene transfer can be performed by introducing a nucleic acid sequence encoding Δ6- and / or 1212-desaturase into cells by methods known in the art. If a transgenic animal is required, pluripotent embryonic stem cells can be prepared with a vector carrying a transgene encoding desaturase and which can develop into an adult animal (USPN 5,162,215; Ono et al. (1996) Comparative Biochemistry and Physidlogy A 113 (3): 287-292; WO 9612793; WO 9606160). In most cases, the transgene will be modified to express a large amount of desaturase while increasing PUFA production. For example, the transgene may be modified by a transcriptional and / or translational regulatory region that functions in avian cells, such as promoters, that direct expression in certain tissues and in parts of eggs such as yolk. Gene regulatory regions can be obtained from various sources, such as chicken anemia virus or avian leukosis virus, or avian genes such as the chicken ovalbumin gene.

• ·

Expression in insect cells

Production of PUFA in insect cells can be performed using baculovirus expression vectors carrying one or more desaturase transgenes. Baculovirus expression vectors are available from several commercial sources such as Clontech. The invention describes methods for producing hybrid and transgenic algae strains, such as seaweed, which contain and express a transgenic desaturase. Transgenic seaweed can be prepared as described in USPN 5,426,040. As in other expression systems, the timing, extent of expression, and activity of the desaturase transgenes can be regulated by linking the polypeptide coding sequence to a suitable transcriptional or translational regulatory region that has been selected for a particular use. Of particular interest is the promoter region which can be induced under preselected growth conditions. For this purpose, the introduction of temperature-sensitive mutations and / or mutations in response to metabolites into sequences encoding the transgene desaturase, its regulatory regions and / or the genome of the cells into which the transgene is introduced can be used.

The transformed host cell is cultured under suitable conditions that have been adjusted to achieve the desired end result. For host cells cultured in culture, conditions are typically optimized to produce the largest and most economical PUFA yield related to the selected desaturase activity. During cultivation it is possible to optimize: carbon source, nitrogen source, substrate addition, final concentration of added substrate, form of added substrate, aerobic or anaerobic growth, culture temperature, induction agent, induction temperature, growth phase on induction, growth phase at harvest, pH, density and maintaining selection. The microorganisms of interest, such as yeast, preferably grow on a selection medium. For the cultivation of yeast, a complex medium such as

is a peptone broth (YPD) or a defined culture medium, such as a minimal medium (containing amino acids, yeast nitrogen base and ammonium sulphate but lacking a selectable component, for example uracil). The substrate was dissolved in ethanol before addition. If necessary, expression of the poloypeptide can be expressed, for example, by adding galactose, thereby inducing expression from the GAL promoter.

Expression in plants

Production of PUFAs in plants can be accomplished using various plant transformation systems such as Agrobacterium tumefaciens, plant viruses, transformation of particles into cells, and the like. All of this is described in US Application Serial Nos. 08 / 834,033 and 08 / 956,985 and the continuation of that application.

Expression in animals

Expression in the cells of the host animal may be temporary or stable. Temporary expression can be accomplished by methods well known in the art, for example, by infection or lipofection, and can be repeated to maintain the desired expression level of the established construct (Ebert, WO 94/05782). Stable expression can be achieved by incorporating the construct into the host genome, resulting in a transgenic animal. The construct can be introduced, for example, by microinjection into the fertilized egg nucleus or by transfection, retroviral infection, or other methods by which the construct can be introduced into a generated cell line that can be introduced into an adult animal (US Patent No. 4,873,191; US Patent No. 5,530,177; U.S. Patent 5,565,362, U.S. Patent No. 5,366,894 to Wilmut et al., (1997) Nature 385: 810). Recombinant eggs or embryos are transferred to a surrogate mother (US Patent No. 4,873,191; US Patent No. 5,530,177; US Patent No. 5,565,362; US Patent No. 5,366,894; Wilmut et al., (1997) Nature 385: 810).

• · »

After birth, transgenic animals are identified by, for example, the presence of an established marker gene, such as fur color, by PCR or Southern blotting of blood, tissue or milk samples to detect the established construct, or immunological or enzymatic tests to detect the expressed protein or produced products (US Patent No. 4,873,191; US Patent No. 5,530,177; US Patent No. 5,565,362; US Patent No. 5,366,894; Wilmut et al., (1997) Nature 385: 810). The resulting transgenic animals may be totally transgenic or may be mosaic having transgenes in a particular set of their cells. The onset of mammalian cloning is accomplished by fusing a nucleotide cell with an enucleated egg. This is followed by transfer to a surrogate mother and it is possible to obtain an animal or cell containing the introduced construct. Before this happens, it is essential that the transgene is present in the germ line of the animal in order to propagate (Wilmut et al., (1997)

Nature 385: 810).

Expression in a host animal has some efficacy, particularly when the animal is a domestic animal. In the case of the production of PUFAs in fluids that can be easily obtained from a host animal, such as milk, the transatur desaturase can be expressed in mammalian cells from the female host and the PUFA content in the host cells can vary. The desaturase transgene can be adapted for expression by remaining in mammalian cells or secreted into milk, then the PUFA production product is found in milk (WO 95/24488). Expression can be targeted to mammalian tissue using specific regulatory sequences such as bovine α-lactalbumin, α-casein, β-casein, γ-casein, κ-casein, β-lactoglobulin or whey acid protein and may also include one or more introns and / or secretion signal sequences (US Patent No. 5,530,177 to Rosen; US Patent No. 5,565,362 to Clark et al .; U.S. Patent No. 5,366,894 to Garner et al .; WO 95/23868).

• ·

The expression of desaturase transgenes or antisense desaturase transcripts that have been adapted in this way can be used to alter the amount of specific PUFAs or derivatives thereof found in animal milk. In addition, desaturase transgene (s) can be expressed either alone or together with another transgene to produce animal milk that contains a higher PUFA component or ratios and concentrations of PUFA that correspond to human breast milk (Prieto et al., WO 95/24494).

such as acylglycerols, glycolipids and may be or their methanol and

Fatty acid cleaning

Desaturated fatty acids may be found in the host microorganism or animal as free fatty acids or in conjunction with forms, phospholipids, sulfolipids, or extracted from the host cell in a variety of ways well known in the art. Such methods may include organic solvent extraction, sonication, supercritical fluid extraction using, for example, carbon dioxide. Further, physical processes such as pressure combinations may be used. Of particular interest is extraction with chloroform. If necessary, the aqueous layer may be acidified to protonate the negatively charged portions and thereby increase the productization of the desired products in the organic layer. After extraction, the organic solvents can be removed by evaporation in a stream of nitrogen. When isolated in conjugated forms, the products can be enzymatically or chemically cleaved to release the free fatty acid or less complex conjugate. These may be subject to further manipulation to produce the desired end product. Conjugated forms of fatty acids are cleaved by potassium hydroxide.

If further purification is necessary, standard methods can be used. Such methods may include extraction, urea application, fractional crystallization, HPLC, fractional

Distillation, silica gel chromatography, high speed centrifugation or distillation or a combination of the above methods. The protection of the reaction groups, such as acidic or alkenyl groups, can be carried out in any step of known methods, for example alkylation or iodization. Methods usually involve methylation of fatty acids to form methyl esters. Similarly, the protecting group can be removed at any step. Purification of fractions containing GLA, SDA, ARA, DHA and EPA can be accomplished by urea application and / or fractional distillation.

Use of fatty acids

There are a number of uses of the fatty acids of the invention. The DNA-based probes of the invention can be used in methods for isolating related molecules or in methods for detecting organisms expressing desaturases. When used as probes, DNA or oligonucleotides must be detectable. This is usually accompanied by capturing the markers either internally, for example by incorporating the treated residue, or at the 5 'or 3' end. Such labeling may be detected directly, may bind a second molecule that is detectably labeled, or may bind to an unlabeled second molecule and a detectably labeled tertiary molecule. This process can be extended, if practical, to achieve a satisfactory detection signal without undesirable background signal levels. Secondary, tertiary e bridging systems may include the use of antibodies against any other molecule, including labeling or other antibodies, or may include any molecules that bind to one another. For example, the biotinstreptavidin / avidin system. Detectable labeling typically includes radioactive isotopes, molecules that chemically or enzymatically produce or alter light, enzymes that produce detectable reaction products, magnetic molecules, fluorescent molecules, or molecules that, upon binding, change their fluorescent or light-emitting characteristics.

directly through measurement

Examples of labeling methods are described in USPN 5,011,770. Alternatively, binding of the target molecules can be detected by changing the temperature of the solution when the probe is isothermal titration calorimetry is coupled, or by coating the probe or target on the surface and detecting a change in light scattering from the surface that occurs by binding the target or probe. This can be done by BIAcore.

PUFAs produced by recombinant methods can be used in various fields. Administration of PUFAs to humans and animals in various forms may lead to increased levels of PUFAs not only by added PUFAs, but also by their metabolic progeny.

Nutritional composition

The invention also provides nutritional compositions. Such compositions for the purposes of the present invention include any food or composition suitable for human use. They can be administered enterally or parenterally. These products upon entry into the body (a) nourish and build tissues or supply energy and / or (b) maintain, restore or promote adequate nutritional status or metabolic function.

The nutritional composition of the invention comprises at least an oil or one acid that is produced in accordance with the invention and may be in solid or liquid form. In addition, the composition may include ingestible macronutrients, vitamins and minerals in an amount necessary for a particular application. The amount of such components varies depending on whether the composition is intended for use by infants, children or adults, for particular reasons such as a metabolic condition (e.g., a metabolic disorder).

Examples of macronutrients that may be added to the composition include, but are not limited to, edible fats, carbohydrates, and proteins. Examples of such edible fats include, but are not limited to, coconut oil, soybean oil, and normal healthy who such supplements are, for example, certain mono- and diglycerides. Examples of such carbohydrates include, but are not limited to, glucose, edible lactose, and hydrolyzed starch. The following are examples of proteins that can be used as a nutritional composition of the invention. These are, for example, soy proteins, electrodialysed whey, electrodialysed skimmed milk, milk whey or hydrolysates of these proteins.

With regard to vitamins and minerals, the following may be added to the nutritional compositions: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and vitamins A, E, D, C and vitamin B complex. In addition, other such minerals and vitamins may be added.

The components used in the nutritional compositions of the invention are of semi-purified or purified origin. The term "semi-purified" or "purified" means a material that has been prepared by purification of natural material or by synthesis.

Examples of nutritional compositions of the invention include, but are not limited to, infant formulas, dietary supplements, and rehydration compositions. The nutritional compositions include, but are not limited to, those employing enteral or parenteral infant supplements, specialty infant formulas, elderly supplements, and p-ro supplements with gastrointestinal problems and / or complaints.

Nutritional composition

A typical nutritional composition of the invention will contain the edible macronutrients, vitamins and minerals in an amount necessary for a particular application. The amount of such substances varies depending on whether the nutrition is intended for administration to normal, healthy individuals who are temporarily exposed to stress or intended for subjects with special needs due to certain chronic or acute stages of the disease (e.g. metabolic disorders). Those skilled in the art will appreciate that the components used in the nutritional compositions of the invention come from a semi-purified or purified source. The term "semi-purified" or "purified" means a material that has been prepared by purification of natural material or by synthesis. These methods are well known in the art (Code of Federal Regulations for Food Ingredients and Food Processing; Recommended Dietary Allowances, 10 ^th Ed., National Academy Press, Washington, DC, 1989).

In a preferred embodiment of the invention, the nutritional compositions are an enteral nutritional product, preferably an enteral nutritional product intended for children or adults. The present invention provides a nutritional composition that is suitable for the nutrition of stressed adults. The nutrient contains, in addition to the PUFAs of the invention, macronutrients, vitamins and minerals in an amount that provides a daily nutritional dose for adult individuals.

Macronutrient components include edible fats, carbohydrates and proteins. Examples of edible fats are coconut oil, soybean oil mono- and diglycerides, and PUFA oils of the invention. Examples of carbohydrates are glucose, edible lactose and hydrolysed corn starch. Typical protein sources are soy protein, electrodialysed whey or electrodialysed nonfat milk or whey, or hydrolysates of said proteins. Other protein sources may be used. These macronutrients may be added in the form of commonly acceptable nutritional substances in an amount corresponding to the composition of human breast milk or the energy base, i.e. the amount referred to is a calorie base.

Methods of making liquid and enteral nutritional formulations are well known in the art and are described in the Examples.

The enteral formulation may be sterilized and subsequently used in a form suitable for direct consumption (RTF) or may be stored in a concentrated solution or as a powder.

• ·

The powder can be prepared by spray-drying the enteral preparation, which is prepared according to the above methods. The preparation may be reconstituted by rehydration of the concentrate. Infant and adult formulas are well known in the art and are commonly available (eg, Similac®, Ensure®, Jevity® and Alimets® from Ross Products Division, Abbott Laboratories). The oil or acid of the invention may be added to any formulation in the amount described below.

The energy density of the nutritional composition when in liquid form may typically be in the range of 0.6 kcal to 3.0 kcal per milliliter. In solid or powder form, the nutritional supplement may contain about 1.2 to more than 9 kcal per gram, preferably 3 to 7 kcal per gram. Generally, the osmolality of the liquid product may be less than 700 mOsm. More preferably, a value of less than 660 mOsm is preferred.

The nutrition typically includes, in addition to the PUFAs of the invention, vitamins and minerals, which helps to receive the required minimum daily dose of these substances. In addition to the above PUFAs, it may be necessary to supplement the nutritional composition with zinc, copper and folic acid and other antioxidants. It is believed that the compounds also provide support to the stressed immune system and thus provide some benefit to the individual. The presence of zinc, copper and folic acid is not necessary to achieve a positive effect on immune suppression. The pharmaceutical composition may also be supplemented with the aforementioned substances.

In a more preferred embodiment, the nutritional composition comprises, in addition to the antioxidant system and the PUFA components, a carbohydrate source wherein at least 5 weight percent of said carbohydrates are an indigestible oligosaccharide. In a more preferred embodiment, the nutritional composition additionally comprises a protein of taurine and carnitine.

The PUFAs or derivatives thereof prepared by the methods described can be used as food substitutes or supplements, particularly in infant formulas in patients nourished intravenously or in the prevention or treatment of malnutrition. Typically, human breast milk has a fatty acid profile comprising about 0.15% to 0.36% DHA, about 0.03% to 0.13% EPA, about 0.30 to 0.88% ARA, about 0.22 % to 0.67% DGLA and about 0.27 to 1.04% GLA. The predominant triglycerides in breast milk are 1,3-diyloyl-2-palmitoyl absorbed than 4,876,107) with 2-palmitoylglycerides, which are better

2-oleoyl or 2-lineoyl glycerides (USPN Then, fatty acids such as ARA, DGLA, GLA and / or EPA prepared according to the invention can be used to change the composition of infant formulas to better mimic the composition of PUFA in human breast milk. for use in pharmacological or dietary supplements, particularly breast milk substitutes or supplements thereof, will preferably contain one or more ARA, DGLA and GLA. More preferably, the oil contains about 0.3 to 30% ARA, about 0, 2 to 30% DGLA and about 0.2 to 30% GLA.

Concentrations and ratios of ARA, DGLA and GLA can be adjusted for certain end uses. When preparing a breast milk supplement or a substitute thereof, the oil composition comprising two or more of ARA, DGLA and GLA is in a ratio of about 1:19:30 to 6: 1: 0.2.

milk in animals shows ARA: DGLA ratio:

ranging from 1:19:30 to 6: 1: 0.2. Preferred ratios are 1: 1: 1, 1: 2: 1 and 1: 1: 4. When the PUFAs are produced together in the host cell, it is desirable to adjust the rate and percent conversion of precursor substrate such as GLA and DGLA to ARA to accurately control PUFA ratios. A 5 to 10% conversion rate of DGLA to ARA can be used to produce an ARA: DGLA ratio of about 1: 9, while a conversion rate of about 75 to 80% can be used at

DGLA 6: 1. Therefore, it can be used as or in the host

For example, maternal DGL that fluctuates in the production of the ARA ratio in an organism's cell culture system, regulating the timing, extent, and specificity of desaturase expression for the purpose of modulating the amount of and PUFA ratio. The oils may be isolated and recombined at the desired concentrations and ratios depending on the expression system used, for example, a cell culture or an animal expressing oil (s) in its milk. The amounts of oils providing the above PUFA ratios can be determined by the following standard protocols. PUFAs or host cells containing them can also be used as animal feed supplements, changing the fatty acid composition of milk or tissues to a composition more acceptable for administration to humans or animals.

In the case of a dietary supplement, PUFAs or derivatives thereof may be incorporated into food oils, fats or margarines which are formulated so that the recipient receives the desired amount in normal use. PUFAs may also be added to infant formulas, nutritional supplements or other food products and may be used as anti-inflammatory or cholesterol lowering agents.

Pharmaceutical compositions

The invention provides a pharmaceutical composition comprising one or more acids and / or the resulting oils produced according to the methods described herein. Such a pharmaceutical composition may contain one or more acids and / or oils as well as a standard, non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle such as phosphate buffered saline, water, ethanol, polyols, vegetable oils, wetting agents or emulsions such as for example a water / oil emulsion. The composition may be in the form of a liquid or a solid. For example, the composition may be in the form of tablets, capsules, an edible liquid or powder, an injectable form, or a topically applied ointment or cream.

Possible routes of administration include, for example, oral, rectal and parenteral administration. The method of administration will of course depend on the desired effect. If the composition is to be used to treat rough, dry or irritated skin or to treat skin or hair affected by the disease, it can be applied topically.

The dose of the composition administered to a patient can be determined by one skilled in the art depending on various factors such as the weight of the patient, the age of the patient, the condition of the immune system.

Depending on the form of the composition, for example, the solution, dispersion, suspension, emulsion or sterile powder may be reconstituted.

The composition of the invention may further be used for cosmetic purposes. They can be added to pre-existing cosmetics. A mixture is formed which can be used as a softening composition.

The pharmaceutical compositions may be used to administer the PUFA component to an individual. In suitable pharmaceutical compositions, they may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders for the preparation of sterile solutions or dispersions. Examples of suitable aqueous or non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Appropriate volatility can be maintained in the case of dispersion, for example, by maintaining the desired particle size and by the use of surfactants. It may also be necessary to use isotonic agents such as sugars, sodium chloride, and the like. In addition to such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and flavoring agents.

The suspensions may contain, in addition to the active compounds, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures thereof, and the like.

* «8« ·

4 / - 9 9 9 9 9 ···

About ··· »· ··

999 99 999 9999

Solid dosage forms such as tablets and capsules can be prepared using methods well known in the art. The PUFAs of the invention may form tablets together with conventional tablet bases such as lactose, sucrose and corn starch in combination with a binder such as acacia, corn starch or gelatin, disintegrating agents such as potato starch or alginic acid and a lubricant such as steric acid or magnesium stearate. Capsules can be prepared by incorporating these excipients into gelatin capsules either alone or with antioxidants and PUFA components. The amounts of antioxidants and PUFA components that should be incorporated into the pharmaceutical composition should be consistent with the above guide.

The term "treat" means either preventing or reducing the occurrence of adverse events. For example, treating immune suppression means either preventing the occurrence of this suppression or reducing the strength of such suppression. The terms "patient" and "individual" are interchangeable and both refer to an animal. The term "animal" means any warm-blooded mammal but is not limited to dogs, humans, monkeys and tailless monkeys. The term "approximately" means an amount that varies within a given range depending on use. Any numeric number or range specified in the specifications could be adjusted according to the above term.

The term "dose" means the amount of a nutritional or pharmaceutical composition administered to a patient in a single application and is designed to introduce an effective amount of antioxidants and structural triglycerides. It will be appreciated by those skilled in the art that a single dose of liquid nutritional powder should provide the aforementioned amounts of antioxidants and PUFAs. The dose should include a volume that a typical adult can consume per administration. This amount can vary widely, depending on the age, weight, sex or medical condition of the patient. Generally, a single dose of a liquid nutritional product would be a liquid nutritional product. The volume should be between 100 and 600 ml, preferably between 125 and 500 ml, and most preferably between 125 and 300 ml.

The PUFAs of the invention may also be added to foods even when dietary supplements are not necessary. For example, the compositions may be added to any type of food that is not limited to margarines, processed butter, cheese, milk, yogurt, chocolate, candies, biscuits, salad oils, fats suitable for heat treatment, meat, fish and beverage.

Pharmaceutical applications

In the case of pharmaceutical use (in human and veterinary medicine), the compositions can generally be administered orally or by any other means that can be successfully absorbed. This means, for example, parenterally (i.e., subcutaneously, into a muscle or intravenously), rectally or vaginally or topically, which may be, for example, a skin ointment or solution. The PUFAs of the invention may be administered alone or in combination with a pharmaceutically acceptable carrier or excipient. When gelatin capsules are available, they become the preferred form of oral administration. Food supplementation as described above can also be performed by the oral route of administration. The unsaturated acids of the invention may be administered in conjugated forms or as salts, esters, amides or pro-forms of fatty acid drugs. The invention provides any pharmaceutically acceptable salts. Sodium, potassium and lithium salts are particularly preferred. Further, Nalkylpolyhydroxamine salts such as N-methylglucamine are described in WO 96/33155. Preferred esters are ethyl esters. As solid salts, the esters can be administered in the form of tablets. For intravenous administration, PUFAs or derivatives thereof may be incorporated into conventional formulations such as Intralipids. The plasma fatty acid profile of a normal adult is 6.64 to 9.46% ARA, 1.45 to 3.11% DGLA, and ·

0.02 to 0.08% GLA. These PUFAs or other metabolic precursors may be administered either alone or in admixture with other PUFAs to achieve a normal fatty acid profile in the patient. If necessary, the individual components of the formulations may be available in the form of kits that are suitable for single or multiple use. A typical dose of a certain fatty acid is 0.1 mg to 20 mg or even 100 g per day, and preferably 10 mg to 1, 2, 5 or 10 g per day or molar equivalent amounts of derivatives thereof. The invention further provides parenteral nutritional compositions comprising about 2 to 30 weight percent fatty acids calculated as triglycerides. Compositions containing about 1 to 25 weight percent of the total PUFA composition as GLA are preferred (USPN 5,196,198). Other vitamins may be added to the composition, and particularly fat-soluble vitamins such as vitamin A, D,

E and L-carnitine. If necessary, preservatives such as α-tocopherol can typically be about 0.1 weight percent.

Suitable pharmaceutical compositions may comprise physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders suitable for reconstitution into sterile injectable solutions or dispersions. Examples of suitable and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol and the like), suitable mixtures thereof (such as olive oil) and injectable such as ethyl oleate. Appropriate fluidity can be maintained in the case of dispersions, for example, by maintaining the desired particle size or by using surfactants. It may also be necessary to include isotonic agents such as sugars, sodium chloride, and the like. In addition to such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

vegetable oils organic esters

The active ingredient suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures thereof.

Particularly preferred compositions comprise diacetyltartaric acid esters of mono- and diglycerides dissolved in an aqueous medium or solvent. Diacetyltartaric acid esters of mono- and diglycerides have an HLB value of about 9-12 and are substantially more hydrophilic than existing antimicrobial lipids having HLB values of 2-4. These existing hydrophobic lipids cannot form in the form of aqueous compositions. These lipids can be dissolved in an aqueous medium in combination with diacetyltartaric acid esters and mono-diglycerides. According to this embodiment, diacetyltartaric acid esters of mono- and diglycerides (e.g. DATEM-C12: 0) are melted with other active antimicrobial lipids (e.g. 18: 2 and 12: 0 monoglycerides) and mixed to form a homogeneous mixture. Homogeneity makes it possible to increase antimicrobial activity. The mixture may be completely dispersed in water. This is not possible without the addition of diacetyltartaric acid esters and mono- and diglycerides. Before adding water, everything is mixed with other monoglycerides. The aqueous composition may then be mixed under sterile conditions with the physiologically acceptable diluents, preservatives, buffers, or propellants required to form the spray or inhalation form.

The invention further provides treatment of a number of disorders with fatty acids. The PUFA supplementation of the invention can be used in the treatment of restenosis after angioplasty. Symptoms of inflammation, rheumatoid arthritis and asthma and psoriasis can be treated with the PUFAs of the invention. Evidence suggests that PUFAs may be involved in calcium metabolism, suggesting that the PUFAs of the invention can be used in the treatment or prevention of osteoporosis and stones in the kidney or urinary tract.

Fatty acids, said cells, and

The PUFAs of the invention may be used in the treatment of cancer. Malignant cells have been shown to exhibit a change in composition. In addition, fatty acids slow growth and cause their death and increase their sensitivity to chemotherapeutic agents. It is further disclosed that GLA causes re-expression of Ekaderin cell adhesion molecules in cancer cells, the loss of expression of said molecules being associated with aggressive metastasis formation. Clinical trials of intravenous application of water-soluble lithium salt of GLA to patients with pancreatic malignancies show a substantial increase in surviving patients. Administration of PUFA is also suitable for the treatment of cachexia associated with malignancy.

The PUFAs of the invention can also be used in the treatment of diabetes (USPN 4,826,877; Horrobin et al., Am. J. Clin. Nutr. Vol. 57 (Suppl.), 732S-737S). Altered fatty acid metabolism and composition is demonstrated in animals suffering from diabetes. These changes suggest that they are involved in the complications of long-term diabetes. These complications include retinopathy, neuropathy, nephropathy and reproductive system damage. Primrose oil, which contains GLA, is able to prevent and repair nerve damage in long-term diabetes.

The PUFAs of the invention may be used in the treatment of eczema, in reducing blood pressure. Fatty acid deficiency is manifested by eczema and studies suggest that GLA has a positive effect on eczema. GLAs are also involved in reducing the high pressure associated with stress. GLA and DGLA have been shown to inhibit platelet aggregation, cause vasodilation, lower cholesterol levels, and inhibit vascular wall and fibrous tissue smooth muscle proliferation (Brenner et al., Adv. Exp. Med. Biol. Vol. 83, p.85-101) , 1976). Administration of GLA or DGLA alone or in combination with EPA reduces or prevents gastrointestinal bleeding and other side effects associated with the administration of non-steroidal anti-inflammatory drugs (USPN 4,666,701). GLA and DGLA also prevent or treat endometriosis and premenstrual syndrome (USPN 4,758,592) and treat myalgic encephalomyelitis and chronic fatigue after viral infection (USPN 5,116,871).

Other uses of the PUFAs of the invention include use in the treatment of AIDS, multiple sclerosis, acute respiratory syndrome, hypertension and inflammatory skin disease. The PUFAs of the invention may also be used in formulations intended for overall enhancement and for geriatric treatment.

Veterinary application

It should be noted that the above pharmaceutical and nutritional compositions can also be administered to animals as well as humans. Animals can have the same needs and the same conditions as humans. The oil or acids of the invention may be used as a supplement to animal feed.

Overview of the drawings

Figure 1 shows the possible pathway for the synthesis of arachidonic acid (20: 4 Δ5, 8, 11, 14) and stearadic acid (18: 4 Δ6, 9, 12, 15) from palmitic acid (C ₁₆ ) from various organisms that include algae, Mortierella and humans. These PUFAs may serve as precursors to other molecules important to humans and other animals. Such molecules include prostacyclins, leukotrienes, and prostaglandins. Some of them are shown here.

Figure 2 shows the possible pathway of PUFA production alongside ARA, including EPA and DHA for various organisms.

Figure 3A-E shows the DNA sequence of Mortierelle alpina A6-desaturase and the deduced amino acid sequence:

Figure 3A-E (SEQ ID NO 1 A6DESATURAZY cDNA).

Figure 3A to E (SEQ ID NO 2 amino acid A6DESATURAZY).

Figure 4 shows the alignment of part of the Mortierella alpina β-desaturase amino acid sequence with other related sequences.

Figure 5A-D shows the DNA sequence of A12-desaturase and the deduced amino acid sequence:

Figure 5A-D (SEQ ID NO 3 cDNA Δ12 DESATURASE)

Picture No. 5A to D (SEQ ID NO 4 amino acid Δ12 DESATURASES). Picture No. 6A to 6B shows effect different expression constructions on expresi GLA . in yeast. Picture no. 7 A a 7B shows the effect host strain on production of GLA. Picture No. 8A a 8B shows the effect host strain on production of GLA in S. cerevisiae strain SC334

Figure 9 shows the alignment of the Ma 29 protein sequence and contig 253538a.

Figure 10 shows the alignment of the Ma 524 protein sequence and contig 253538a.

Sequence description:

SEQ ID NO: 1 shows the DNA sequence of β-desaturase

Mortierella alpina.

SEQ ID NO: 2 shows the protein sequence of Mortierella alpina β-desaturase.

SEQ ID NO: 3 shows the DNA sequence of A12-desaturase

Mortierella alpina.

SEQ ID NO: 4 shows the protein sequence of Mortierella alpina Al2-desaturase.

SEQ ID NOs: 5-11 show different desaturase sequences.

SEQ ID NO: 11 and SEQ ID NO: 12 show the amino acid motifs of the desaturase sequence.

SEQ ID NOS: 13-18 show sequences of different PCR primers.

SEQ ID NO: 19 and SEQ ID NO: 20 show the nucleotide and amino acid sequence of Dictyostelium discoideum desaturase.

SEQ ID NO: 21 and SEQ ID NO: 22 show the nucleotide and amino acid sequences of Phaeodactylum tricornutum desaturase.

SEQ ID NOS: 23-26 shows the nucleotide and deduced amino acid sequence of the Schizochytrium cDNA clone.

SEQ ID NOS: 27-33 shows the nucleotide sequence for human desaturases.

SEQ ID NO: 34 to SEQ ID NO: 40 depict the peptide sequences of human desaturases.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Construction of a Mortierella alpina cDNA library

Total RNA was isolated from three day cultures of Mortierella alpina producing PUFA using the protocol of Hoge et al. (1982) Experimental Mycology 6: 225-232. RNA was used to prepare double stranded cDNA using a lambda-ZipLox system from BLR, following the manufacturer's instructions. Separately, several M. alpina cDNA fractions of different sizes were packaged, resulting in libraries with different average size inserts. The full-length insert library contains approximately 3 x 10 ⁶ clones with an average insert size of 1.77 kb. The sequencing library contains approximately 6 1.1 kb in size.

x 10 ⁵ clones with an insert of average

Example 2: Isolation of the 66-desaturase nucleotide sequence from Mortierella alpina

By random sequencing of clones from the M. alpina cDNA library suitable for the sequencing described in Example 1, a nucleic acid sequence was obtained from the Ma524 partial clone cDNA that encodes a β-desaturase fatty acid. The plasmids containing the cDNA were excised as follows:

pg of phage was combined with 100 µΐ of an E. coliDHlOB (ZIP) culture that was cultured on ECLB culture medium with 10 µg / ml kanamycin, 0.2% maltose and 10 mM MgSO 4 and incubated at 37 ° C for 15 minutes . 0.9 ml of SOC was added and 100 μΐ of bacteria was immediately applied to each plate containing 10 ECLB + 50 µg Pen. A 45 minute time to memorize the culture was not necessary. The plates were incubated overnight at 37 ° C. Colonies were plated in ECLB + 50 µg Pen culture medium and incubated overnight. Giycerol stock solutions were prepared from this culture and mini DNA isolation was performed. An aliquot of the culture used as mini isolation was stored as a stock solution with giycerol. Applying the culture to plates with ECLB + 50 pg Pen / ml culture medium resulted in more colonies and a larger portion of the colonies contained inserts compared to plates containing 100 pg / ml Pen.

Colonies were randomized and plasmid DNA was isolated using a mini isolation kit. The DNA sequence was obtained from the 5 'end of the cDNA insert and compared from the National Center for Biotechnology databases using the BLASTX algorithm. Ma524 putative desaturase based on previously identified desaturases.

A full-length cDNA clone was isolated from the M. alpina library and designated pCGN5532. The CDNA is present in the form of a 1617 bp insert in the vector pZL1 (BRL) a

Information (NCBI) was identified as sequence homology

DNA starts with ATG, amino acids. It is known that conserved frames contain an open reading frame encoding 457 that three conserved "histidine" membrane-bound desaturases (Okuley, et al. (1994) The Plant Cell 6: 147-158) are present at amino acid positions 172 to 176, 209 to 20, respectively. 213 and 359 to 399 (shown in Figure 3). As with other váz655 membrane-bound desaturases, the final motif of the HXXHH histidine frame is QXXHH. The amino acid sequence of Ma524 shows substantial homology with part of the Caenorhabd.itis elegans WO6D2.4 cosmid, with the cytochom b5 / sunflower desaturase fusion protein and with β-desaturases from Synechocystis and Spirulina. In addition, Ma524 shows homology to the borage desaturase Δ6 desaturase amino acid sequence (PCT WO 96/21022). Ma524 thus encodes β-desaturase, which is related to β-desaturase of borage and algae. The peptide sequences are set forth in SEQ ID NOs: 5-11.

The N-terminus of the encoded protein was found to exhibit substantial homology to cytochrome b5 proteins. The Mortierella cDNA clone represents a fusion between cytochrome b5 and fatty acid desaturase. Since cytochrome b5 is believed to function as an electron donor for membrane-bound desaturase enzymes, it is possible that the N-terminal region of cytochrome b5 of this desaturase protein is involved in this function. This may be advantageous when the desaturase is expressed in heterogeneous systems to produce PUFA. However, it should be noted that although the amino acid sequences of Ma524 and β-desaturase contain regions of homology, the composition of the cDNA bases is substantially different. For example, borage cDNA has been shown to contain 60% A / T, some regions contain 70% A / T, while Ma524 shows an average of 44% A / T, with no region exceeding 60%. This may cause cDNA expression in microorganisms or in animals that favor a different base composition. It is known that poor expression of recombinant genes can occur when the host prefers a composition that is different from that of the introduced gene. Mechanisms of such poor expression include reduced stability, hidden splice sites and / or translation of mRNA, and the like.

Example 3: Identification of ββ-desaturases homologous to β6 desaturase of Mortierella alpina

Nucleic acid sequences that code for putative 66-desaturases were identified using the BLASTX algortime database &quot; Expressed Sequence Tag (&quot; EST) by NCBI using the Ma524 amino acid sequence. Several sequences show substantial homology. In particular, the deduced amino acid sequence of the two Arabidopsis thaliana sequences (deposit numbers F13728 and T42806) show homology to two different regions of the deduced amino acid sequence Ma524. The following primers were created:

ATTS4723-FOR (complementary to F13728) SEQ ID NO: 13

5 'CUACUACUACUAGGAGTCCTCTACGGTGTTTTG a

T42806-REV (complementary to T42806) of SEQ ID NO: 14

5 'CAUCAUCAUCAUCAUATGATGCTCAAGCTGAAACTG.

Five total RNA programs isolated from Arabidopsis thaliana were reverse transcribed using BRL Superscript Rtase and TSyn primer (5'CCAAGCTTCTGCAGGAGCTCTTTTTTTTTTTTTTT-3 ') and labeled as SEQ ID NO: 12. The PCR was performed at 50 μΐ where: template obtained from 25 ng of total RNA, 2 pM of each primer, 200 μΜ of deoxyribonucleotide triphosphate, 60 mM Tris-Cl, pH 8.5, 15 mM (NH4) 2SO4, 2 mM MgCl2, 0.2 units of Taq polymerase. The conditions in the thermocycler were as follows: 94 ° C for both 30 seconds, 50 ° C for 30 seconds, 72 ° C for 30 seconds. PCR was continued for 35 cycles, followed by further extension at 72 ° C for 7 minutes. The result of the PCR was an approximately 750 bp fragment that was subcloned, designated 12-5, and sequenced. Each end of this fragment was designed to correspond to the EST Arabidopsis from which the PCR primers were generated. The putative amino acid sequence 12-5 was compared to the Ma524 and EST sequences from human (W28140), mouse (W53753) and C.elegans (R05219) (Figure 4). Mortirella β-desaturase homology patterns indicate that these sequences represent putative desaturase polypeptides. Based on these experiments is

it is likely that full-length genes can be cloned using EST-based probes. This is followed by cloning, whereby the genes can then be inserted into expression vectors and expressed in host cells and their specific activity of β-desaturase or other desaturase can be determined as described below.

Example 4: Isolation of the A12-desaturase nucleotide sequence from Mortierella alpina

Based on the accumulation of fatty acids, it appears likely that Mortierella alpina has ωβ-type desaturase. ωβdesaturase is responsible for the production of linolenic acid (18: 2) from oleic acid (18: 1). Linolenic acid (18: 2) is a substrate for β-desaturases. This experiment was designed to determine whether Mortierella alpina has an 1212-desaturase polypeptide. If so, the corresponding nucleotide sequence was determined.

A random colony from a library suitable for M. alpina Ma648 sequencing was sequenced and identified as a putative desaturase based on DNA sequence homology with previously identified desaturases as described for Ma524 (Example 2). The nucleotide sequence is shown in SEQ ID NO: 13. The peptide sequence is shown in SEQ ID NO: 4. The deduced amino acid sequence from the 5 'end of the Ma648 cDNA shows substantial homology to microsomal soybean ωβ (Δ12) desaturase (deposit # L43921) as well as with bean oleate hydroxylase (deposit # U22378). When compared to other fatty acid desaturase sequences ωβ (Δ12) and ω3 (Δ15), some homology was also observed.

Example 5: Expression of M. alpina desaturase clones in baker's yeast

Transformation of yeasts · · · · * * * * kv kv kv kv kv kv kv • kv kv kv kv kv • kv kv

Transformation of yeast with lithium acetate was performed according to standard protocols (Methods in Enzymology, Vol. 194, p. 186-187, 1991). The yeast was cultured in culture medium

YPD at 30 ° C. The cells were centrifuged, resuspended in TE, and again centrifuged and resuspended in TE containing 100 mM lithium acetate. After centrifugation, the cells were resuspended in TE / lithium acetate. Resuspended yeast was incubated at 30 ° C for 60 minutes with stirring. Carrier DNA was added and aliquots of yeast culture were prepared in tubes. Next, transforming DNA was added to the tubes and incubated for 30 minutes at 30 ° C. PEG solution (35% (w / v) PEG 4000, 100 mM lithium acetate, TE pH 7.5) was added, followed by incubation for 50 minutes at 30 ° C.

A thermal shock was performed at 42 ° C for 5 minutes, the cells were centrifuged into a pellet, washed with TE, centrifuged again and resuspended in TE. The resuspended cells were then plated on selective medium plates.

Desaturase expression in transformed yeast

Mortierella alpina cDNA clones were tested for desaturase activity in baker's yeast. A15-canola desaturase (obtained by PCR using the first strand cDNA from Brassica napus cultivar 212/86 seeds using published sequence primers (Arondel et al. Science 258: 1353-1355)) was used as a positive control.

The 1515-desaturase gene and the gene from the cDNA clones Ma524 and Ma648 were inserted into the expression vector pYES2 (Invitrogen) to generate plasmids pCGR-2, pCGR-5 and pCGR-7. These plasmids were transfected into yeast S. cerevisiae strain 334 and expressed upon galactose induction in the presence of substrates that allow detection of specific desaturase activity. The control strain was S. cerevisiae strain 334 containing the unchanged vector pYES2. The substrates used, the products produced and indicated on ARA indicate (conversion to GLA conversion to ALA indicates desaturase activities were: DGLA (conversion of des5-desaturase activity), linolenic acid indicates β-desaturase activity, A15-desaturase activity), oleic acid (endogenous substrate) prepared by S. cerevisiae yeast, conversion to linolenic acid indicates A12-desaturase activity that is missing from S. cerevisiae yeast) or ARA (conversion to EPA indicates A17-desaturase activity).

The cultures were cultured for 48 to 52 hours at 15 ° C. The cells were centrifuged, the pellet was washed with sterile ddH ₂ O, and the pellet was again prepared by centrifugation. The pellets were suspended by vortexing in methanol. Chloroform with tritridecanone (as an inert standard) was added. The mixtures were incubated for at least one hour at room temperature or overnight at 4 ° C. The chloroform layer was extracted and filtered using a Whatmance filter with one gram of anhydrous sodium sulfate to remove particulates! and the rest of the water. The organic solvents were evaporated at 40 ° C under a stream of nitrogen. The extracted lipids were then derivatized to fatty acid methyl esters (FAME) and analyzed by chromatography (GC) by adding 2 mL of 0.5 N potassium hydroxide in methanol. The tube was sealed. The samples were heated to 95-100 ° C for 30 minutes and cooled to room temperature. Then approximately 2 ml of 14% boron trifluoride in methanol was added and the heating was repeated. After the extracted lipid mixture was cooled, 2 mL of water and 1 mL of hexane were added to extract FAME for GC analysis. The percent conversion was calculated by dividing the product produced by the sum of the product produced and the substrate added and then multiplied by 100. To calculate the oleic acid conversion percentage where no substrate was added, linolenic is divided by the total oleic acid produced and the product produced.

Linolenic acid multiplied by 100. Desaturase activity results are shown in Table 1 below.

Table 1

M. alpina desaturase expression in baker's yeast

Clone enzymatic activity % substrate conversion pCGR-2 A15-desaturase kanoly Δ6 0 (18: 2 to 18: 3ω6) Δ15 16.3 (18: 2 to 18: 3co3) Δ5 2.0 (20: 3 to 18: 3ω3) Δ17 2.8 (20: 4 to 20: 5ω3) Δ12 1.8 (18: 1 to 18: 2ω6) pCGR-5 (M. alpina Ma524) Δ6 6, 0 Δ15 0 Δ5 2.1 Δ17 0 Δ12 3.3 PCGR-7 (M. alpina Ma648) Δ6 0 Δ15 3.8 Δ5 2.2 Δ17 0 Δ12 63.4

A15-desaturase control clone shows 16.3% substrate conversions. The pCGR-5 clone expressing the Ma524 cDNA converted 6% of the substrate to GLA, indicating that the gene encodes β-desaturase. The background (non-specific substrate conversion) in this case is between 0 and 3%. Furthermore, the substrate was found to inhibit activity at various concentrations. When the substrate was added at a concentration of 100 μΜ, the conversion percentage decreased compared to the substrate concentration of 25 μΜ (see below).

• ·

In addition, if the substrate concentrations range from about 5 μΜ to 200 μΜ, the conversion percentage is about 5 to 75% or greater. These data show that desaturases with different substrate specificities can be expressed in a heterogeneous system and are used to produce long chain polyunsaturated fatty acids.

Table 2 represents interesting fatty acids as a percentage of total lipids extractable from the yeast host S. cerevisiae 334 with the indicated plasmid. No glucose is present in the growth medium. Affinity gas chromatography was used to separate used to verify the identity of the product (s). A15-desaturase of B. napus, resulting in lipids. The expected product is GC / MS and linolenic acid was detected when its linolenic acid substrate was added exogenously to the induced yeast culture. This finding suggests that expression of the desaturase gene in yeast produces a functional enzyme and a detectable amount of product under current growth conditions. Both exogenously added substrates were absorbed by the yeast, although slightly less long chain PUFA, which is dihomo-γ-linolenic acid (20: 3), was incorporated into the yeast, rather than linolenic acid (18: 2) when added in free form. to induce a yeast culture. Γ-linolenic acid was detected in yeast as new PUFA when during induction and expression in S. cerevisiae 334 (pCGR-5). acid was present This indicates that PUFAs exhibit aktivitu6 activity from pCGR-5 (MA524). Linolenic acid was identified in extracted lipids derived from the expression of S. cerevisiae 334 (pCGR-7), the MA648 cDNA from M. alpina was classified as A12-desaturase linolenic. desaturases • ·

acid expressed as a percentage of total lipids extracted from yeast

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Example 6: Optimization of culture conditions

Table 3A shows the effect of substrate concentration of exogenous free fatty acids on the uptake and conversion of the fatty acid product in yeast. It is expressed as a percentage of the total yeast lipid extracted. In all cases, a small amount of exogenous substrate (1 to 10 μΜ) results in the absorption of a small amount of the fatty acid substrate and formation of the product. Concentrations of free fatty acids between 25 and 50 μΜ in culture and induction media provide the highest percentage of fatty acid product formed, while concentrations of 100 μΜ and subsequent high uptake appear to reduce or inhibit desaturation activity. The amount of fatty acid substrate suitable for yeast expressing 1212-desaturase is similar under the same culture conditions because the substrate, oleic acid, is an endogenous yeast fatty acid. The use of α-linolenic acid as an additional substrate for pCGR-5 (Δ6) produces the expected product stearidonic acid (Table 2).

3).

Table 3B illustrates well the feedback inhibition of a substrate with a high fatty acid concentration, comparing the range of percent conversion of fatty acid substrates to their products. In all cases, the substrate concentration of 100 μΜ in the culture medium reduces the percentage conversion to product. Absorption of α-linolenic acid is comparable to other PUFAs added in free form, while the percentage of conversion of Á6-desaturase 3.8 to 17.5% to stearidonic acid product is the lowest of all substrates tested (Table 3B). Effect

A culture medium such as YPD (rich culture medium) versus minimal glucose-containing culture medium to A12-desaturase conversion rate is dramatic.

Not only did the conversion rate of oleic acid to linolenic acid (Table 3B) decrease for the cultivation and induction of Δ12desaturase expression in yeast (Table 3B), but the percentage of linolenic acid formed also decreased by 11% (Table 3).

3A). The effect of the culture medium composition is also evident when glucose is present in the kultiv6 desaturase culture medium, since the percentage of substrate absorption was reduced to a concentration of 25 μΜ (Table 3A). However, the conversion rate remains the same and the percentage of product formed decreases as the percentage of glucose decreases.

Table no. Of incorporated extracts

3A: Effect of substrates and added substrate product constituted by percentage in yeast

Plasmid v pCGR-2 PcGR-5 pCGR-5 pCGR-7 yeast (Δ15) (Δ6) (Δ6) (Δ12) Substrate / product 18: 2 / a- 18: 3 18: 2 / γ- 18: 3 and- 18: 3/18: 4 18: 1 * / 18: 2 1 μΜ sub. ND 0.9 / 0.7 ND ND 10 μΜ sub. ND 4.2 / 2.4 10.4 / 2.2 ND 25 μΜ sub. ND 11 / 3.7 18.2 / 2.7 ND 2 5 μΜΟ sub. 36.6 / 7.20 25.1 / 10.30 ND 6.6 / 15.80 50 μΜ sub. 53.1 / 6.50 ND 36,2 / 3 10.8 / 13 + 100 μΜ sub. 60.1 / 5.70 62,4 / 40 47.7 / 1.9 10 / 24.8

Table 3B: Effect of substrate concentration in culture medium on percentage conversion of fatty acid substrate to product in yeast extracts.

Plasmid v yeast pCGR-2 (Δ15) pCGR-5 (Δ6) pCGR-5 (Δ6) pCGR-7 (Δ12) Substrate -> product 18: 2 / a-18: 3 18: 2 / γ — 18: 3 o-18: 3/18: 4 18: 1 * / 18: 2

1 μΜ sub. ND 43.8 ND ND 10 μΜ sub. ND 36, 4 17.5 ND 25 μΜ sub. ND 25.2 12.9 ND 25 μΜΰ sub. 16, 4D 29, ID ND 70.50 50 μΜ sub. 10, 9D ND 7.7 54, 6 + 100 μΜ sub. 8,7D 60 3.8 71.3

□ glucose is not present in culture medium + yeast peptode broth (YPD) * 18: 1 is an endogenous yeast lipid sub. the substrate substrate concentration ND was not performed

Table 4 shows the amount of fatty acid produced by recombinant desaturase from induced yeast cultures when different amounts of free fatty acid substrate were used. Fatty acid weight was determined because the total amount of lipids varies dramatically when growth conditions, such as the presence of glucose in yeast culture and induction media, change. In order to better determine the conditions where recombinant desaturase will produce the maximum PUFA product, a large number of individual fatty acids have been tested. The absence of glucose in the culture medium reduces the amount of linolenic acid produced by 1212-desaturase. In the case of A12-desaturase, the amount of total yeast lipids decreased by almost half in the absence of glucose. Conversely, when glucose is present in yeast growth medium suitable for -6-desaturase, γ-linolenic acid production has decreased by almost half, while the total amount of yeast lipid produced does not change with the presence / absence of glucose. It is possible that glucose serves as a modulator of A66-desaturase activity.

Table 4: Fatty acid produced from yeast extracts expressed in pg.

Plasmid in yeast (enzyme) pCGR-5 (Δ6) pCGR-5 (Δ6) pCGR-7 (Δ6) product γ-18: 2 18: 4 18: 2 * 1 μΜ sub. 1.9 ND ND 10 μΜ sub. 5.3 4.4 ND 25 μΜ sub. 10.3 8.7 115.7 25 pMC sub. 29.6 ND 39Π

□ glucose is not contained in culture medium * 18: 1 substrate is endogenous yeast lipid sub. is the substrate concentration

ND has not been implemented

Example 7: Distribution of PUFA in yeast lipid fractions

Table 5 shows the uptake of free fatty acids and their novel products formed in yeast lipids, which are divided into major lipid fractions. Total lipid extract was prepared as described above. The lipid extract was separated on TLC plates and fractions were identified by comparison with standards. The stripes were collected by scraping and internal standards added. The fractions were then treated with detergent and methylated as above and subjected to gas chromatography. The gas chromatograph calculated the amount of fatty acid by comparison with the standard. The phospholipid fraction contains the highest amount of substrate and PUFA product suitable for Δ6-desaturase activity. Obviously, substrates are acceptable for desaturases in phospholipid form.

Table 5. Fatty acid distribution in various yeast lipid fractions expressed in pg »·

Fractions fatty acid phospholipid diglyceride Free oily acid triglyceride Esther cholesterol SC (pCGR-4) Substrate 18: 2 166, 1 6.2 15 Dec 18.2 15, 6 SC (pCGR-4) Product γ- 18: 3 61.7 1, 6 4.2 5, 9 1,2

SC = S. cerevisiae (plasmid)

Example 8: Further optimization of Δ6- and Δ12desaturase culture and coexpression

Growth and induction conditions of optimal desaturase activity in Saccharomyces cerevisiae were evaluated. A strain of Saccharomyces cerevisiae (SC334) capable of producing γ-linolenic acid (GLA) was developed to estimate the possibility of producing PUFA in yeast. The Δ6- and 1212-desaturase genes from M. alpina were co-expressed in SC334. Δ12desaturase expression converted oleic acid (present in yeast) to linolenic acid. Linolenic acid was used as a substrate for β-desaturase in the production of GLA. The amount of GLA produced is in the range of 5 to 8% of the total fatty acids produced in SC334 cultures and the rate of conversion of linolenic acid to γ-linolenic acid is in the range of 30 to 50%. The temperature induction was optimized, and the effect of changing the host strain and upstream promoter sequence on the expression of a6- and A12-desaturase genes (MA524 and MA648) was also determined.

Construction of plasmids

Cloning of pCGR5 as well as pCGR7 is described above. To construct pCGR9a and pCGR9b, the Δ6- and Δ12desaturase genes were amplified using the following primer set. Primers pRDS1 and 3 have restriction sites XhoI and primers pRDS2 and 4 have restriction sites XbaI (indicated in bold). These primer sequences are presented as SEQ ID NOs: 15-18.

I. amplification primers of β-desaturase

pRDS1 TAC CAA GTC GAG AAA ATG GCT GCT GC CCC AGT GTG AGG

pRDS2 AAC TGA TCT AGA TTA CTG CGC

II. A12-desaturase amplification primers

pRDS3 TAC CAA ATC GAT AAA ATG ATC GAT ATC GAT

pRDS4 AAC TGA TCT AGA TTA CTT GAA GAC CAC GTC TCC

PCGR5 and pCGR7 constructs were used as template DNA for amplification of Δ6- and A12-desaturase genes. The amplified products were digested with restriction enzymes XbaI and XhoI to form coherent ends. PCR amplified β-desaturase with XhoI-XbaI clones cloned into pCGR7 was also digested with XhoI-XbaI restriction enzymes. This procedure placed β-desaturase after Δ12 desaturase, with expression driven by the inducible GAL1 protmotor. This construct was designated pCGR9a. Similar to the construction of pCGR9b, the 1212-desaturase with XhoI-XbaI ends was cloned into the XhoI-XbaI restriction sites of pCGR5. In pCGR9b Δ12desaturase was behind the β-desaturase gene far from the GAL promoter.

To construct pCGR10, the pRS425 vector containing the constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was digested with BamHI and pCGR5 was digested with BamHI-XhoI to release the β-desaturase gene. This BamHI digested β-desaturase and pRS425 fragment was filled using Klenow polymerase to form blunt ends. Both fragments were ligated to give pCGR10a and pCGR10b containing the β-desaturase gene in sense and antisense orientation. To construct pCGR11 and pCGR12, Δ6- and Δ12desaturase genes were isolated from pCGR5 and pCGR7 using EcoRI-XhoI double digestion. EcoRI-XhoI fragment6- and 1212-desaturase fragments were cloned into the EcoRI-XhoI digested pYX242 vector. The pYX242 vector has a TP1 promoter (yeast gene) that allows constitutive expression.

Transformation and expression in yeast

Various combinations of pCGR5, pCGR7, pCGR9a, pCGR9b, pCGR10a, pCGR11 and pCGR12 were introduced into different Saccharomyces cerevisiae host strains. Transformation was performed according to the PEG / LiAc protocol (Methods in Enzymology Vol. 194 (1991): 186-187).

The transformants were selected by plating onto plates containing a synthetic medium lacking the appropriate amino acid. pCGR5, pCGR7, pCGR9a and pCGR9b can be isolated on uracil-free selection medium. PCGR10, pCGR11, and pCGR12 constructs can be isolated on a leucine-free selection medium. Cultivation of the cultures and analysis of fatty acids were performed as described in Example 5 above.

Produced by GLA

GLA production requires the expression of two enzymes (Δ6- and Δ12desaturase), which is not present in yeast. In order to express these enzymes at optimal potency, the following constructs or combinations thereof were incorporated into different host strains:

1) pCGR9a / SC334

2) pCGR9b / SC334

3) pCGR10a and pCGR7 / SC334

4) pCGR11 and pCGR7 / SC334

5) pCGR12 and pCGR5 / SC334

6) pCGR10a and pCGR7 / DBY746

7) pCGR10a and pCGR7 / DBY746

The pCGR9 construct contains both Δ6- and 1212-desaturase genes that drive the inducible GAL promoter. SC334 host cells transformed with this construct show no accumulation of total fatty acids (Figure 6A and B, lane 1). However, when the Δ6- and A12-desaturase genes are individually controlled by the GAL promoter, the control constructs are able to express Δ6- and 1212-desaturase, as is evident from the conversion of their substrate to products. The 1212-desaturase gene in pCGR9a is expressed as shown by the 18: 1ω9 to 18: 2ω6 conversion in pCGR9a / SC334, while the β-desaturase gene is not expressed / inactive because 18: 2ω6 does not change to 18: 3ω6 (Figure 4). 6A and B, lane 1).

The construction of pCGR9b also includes genes of both desaturases that are under the control of the GAL promoter, but this is in reverse order compared to pCGR9a. In this case, very small amounts of GLA (<1%) can be detected in pCGR9b / SC334 cultures. Δ12desaturase expression is also very poor, as evidenced by the low 18: 2ω6 percentage in total fatty acids (Figure 6A and B, lane 1).

To test whether expression of both enzymes is under the control of independent promoters enhances GLA production, the β-desaturase gene was cloned into the pRS425 vector. The construction of pCGR10α contains the gene and expression is controlled

PCGR1Ob contains the Δ6a gene and serves as negative

Β-desaturases in the correct orientation, constitutive promoter of GPD. desaturases in opposite orientation control. PCGR10a / SC334 cells produced significantly higher levels of GLA (5% total fatty acids, Figure 6, lane 3) compared to pCGR9a. The Δ6- and 1212-desaturase genes were expressed in large quantities because the conversion of 18: 1ω9-> 18: 2ω6 was 65%, while the conversion of 18: 2ω6-> 18: 3ω6 (β-desaturase) was 30% (Figure 6). , lane 3). As expected, the negative control of pCGR10b / SC334 showed no GLA production.

• · · · ·

In order to optimize GLA production, the a6- and des12desaturase genes were introduced into the pYX242 vector to produce pCGR11 and pCGR12. The vector pYX242 allows for constitutive expression by the TP1 promoter (Alber, T. and Kawasaki, G (1982). J. Mol. And Appl. Genetics 1: 419). The introduction of pCGR11 and pCGR7 into SC334 resulted in the production of 8% GLA with total SC334 fatty acids.

The conversion rates of 18: 1ω9—> 18: 2ω6 and 18: 2ω6—> 18: 3ω6 were approximately 50% and 44% (Figure 6A and B, lane 4). The presence of pCGR12 and pCGR5 in SC334 resulted in 6-6% GLA in total fatty acids with a conversion rate of approximately 50% in the case of

18: 1ω9 at 18: 2ω6 and 18: 2ω6 at 18: 3ω6 (Figure 6A and B, lane 5). Thus, although the amount of GLA in total fatty acids is higher in the combination of pCGR11 / pCGR7 constructs, the rate of substrate to product conversion was better for the pCGR12 / pCGR5 combination.

To determine whether the host strain would increase GLA production, pCGR10α and pCGR7 were introduced into the host strains BJ1995 and DBY746 (obtained from the Yeast Genetic Stock Center, 1021 Donner Laboratory, Berkeley, CA 94720. The genotype of the strain, not DBY746, is Mata, his3-A1, leu2-3, leu2-112, ura3-32, trpl-289, gal). The results are shown in Figure 7. The change of the host strain to BJ1995 did not demonstrate GAL production because the amount of GLA was only 1.31% of total fatty acids and the conversion rate of 18: 1ω9 to 18: 2ωβ was approximately 17% in the BJ1995 strain. No GLA production was observed in DBY746 strain and the conversion of 18: 1ω9 to 18: 2ω6 was very low (less than 1% in the control strain), suggesting that the cofactor required for A12-desaturase expression may not be present in DB746 (Figure 4). 7, lane 2).

To determine the effect of temperature on GLA production, SC334 cultures containing pCGR10α and pCGR7 were cultured at 15 ° C and 30 ° C. The greatest amount of GLA was found in cultures cultured and induced at 15 ° C, as compared to cultures growing at 30 ° C (4.23% vs. 1.68%). This was due to a lower conversion rate of 18: 2ω6 to 18: 3ω6 at 30 ° C (11.6% versus 29% at 15 ° C), despite a higher conversion of 18: 1ω9 to 18: 2ωβ (65% versus 60% at at 30 ° C (Figure 8) These results suggest that Δ12- and β-desaturases may exhibit different optimal expression temperatures.

Of the various experiments tested, the most important influence on desaturase expression was culture temperature, yeast host strain and culture medium components, while the timing of substrate addition and inducer concentration did not significantly affect desaturase expression.

These data show that two DNAs encoding desaturases that can convert LA to GLA or oleic acid to LA can be isolated from Mortierella alpina and can be expressed either singly or in combination in a heterogeneous system and used to produce polyunsaturated fatty acids with long chain. An example is the production of GLA from oleic acid by expressing Δ12- and Δ6desaturase in yeast.

Example 9: Identification of homologues with alp5- and δδ-desaturase of M. alpina

A nucleic acid sequence that encodes a putative des5 desaturase was identified by screening the TBLASTIN expression sequence of the tag databases via NCBI, using amino acids 100-446 of the Ma29 clone as a query. A truncated portion of the Ma29 sequence was used to prevent homology based on a portion of cytochrome b5 at the N-terminus of the desaturase. The deduced amino acid sequence derived from Dictyostelium discoideum (deposit # C25549) shows very substantial homology to Ma29. It also exhibits less but still substantial homology to Ma524. The DNA sequence is presented as SEQ ID NO:

• ·

19. The amino acid sequence was presented as SEQ ID NO:

20 May

Example 10: Identification of alp5- and β-homologues of M. alpina in other PUFA producing organisms.

To find desaturases involved in PUFA production, a cDNA library was constructed from total RNA isolated from Phaeodactylum tricornutum. A plasmid-based cDNA library was constructed in pSPORT1 (GIBCO-BRL) according to the manufacturer's instructions using a commercially available kit (GIBCO-BRL). Random cDNA clones were sequenced and nucleic acid sequences that encode a putative Δ5- or β-desaturase were identified by screening the BLAST databases and comparing the Ma29 and Ma524 sequences. One clone with homology to Ma29 and Ma524 was identified from the Phaeodactylum library, named 144-011-B12. The DNA sequence is present as SEQ ID NO: 21. The amino acid sequence is present as SEQ ID NO: 22.

Example 11: Identification of alp5- and β-homologues of M. alpina in other PUFA-producing organisms.

To find desaturases involved in PUFA production from total RNA isolated from Schizochytrium sp. A cDNA library was constructed. A plasmid-based cDNA library was constructed in pSPORT1 (GIBCO-BRL) according to the manufacturer's instructions using a commercially available kit (GIBCO-BRL). Random cDNA clones were sequenced and nucleic acid sequences that encode a putative Δ5- or β-desaturase were identified by screening the BLAST databases and comparing the Ma29 and Ma524 sequences.

From the Schizochytrium library, one clone with homology to Ma29 and Ma524 was identified, termed 81-23-C7. This clone contains an insert of approximately 1 kb. A partial sequence was obtained from each end of the clone using universal forward and reverse sequencing primers. The DNA sequence of the forward primer is present as SEQ ID NO: 23. The peptide sequence is present as SEQ ID NO: 24. The DNA sequence from the reverse primer is present as SEQ ID NO: 24. The DNA sequence from the reverse primer is present as SEQ ID NO: 25. The amino acid sequence from the reverse primer is present as SEQ ID NO: 26.

Example 12: Human Desaturase Gene Sequence

Based on the homology between the human cDNA sequences and the Mortierella alpina desaturase gene sequences, the human desaturase gene sequence was potentially included in the long chain biosynthesis of polyunsaturated fatty acids. which

Three conserved histidine boxes, with vi, are found to be conserved among membrane bound desaturases. As with other membrane-bound desaturases, the final motif of the HXXHH histidine box is QXXHH. The amino acid sequence of putative human desaturases shows homology to Δ5, Δ6 and 1212-desaturases.

The Δ5- and β-desaturase cDNA sequences were used to search the LifeSeq database of Incyte Pharmaceuticals, Inc, Palo Alto, California 94304. The A5-desaturase sequence was split into fragments 1) amino acid # 1 to 150, 2) amino acid # 151 to 300 and 3) amino acid numbers 301 to 446. The β-desaturase sequence was divided into three fragments 1) amino acid numbers 1 to 150, 2) amino acid numbers 151 to 300, and 3) amino acid numbers 301 to 457. examined against the database using the tblastn algorithm for the protein sequence detabase dynamically translated into all six reading frames (both chains).

The M. alpina Δ5- and β-desaturase polypeptide fragments 2 and 3 exhibit homology to the CloneID sequences as shown in Table 6. The CloneID represents each of the sequenced sequences.

This algorithm for nucleotide sequences from the Incyte LifeSeq database. After the results were published, the "tblast information of the clones was searched under conditions of stringency> / = 50 and the product score was </ = 100 at different CloneiD numbers. The Information Results screen provides information including ClusterID, Cloneid, Library, HitID, Hit description. The ClusterID number shows information about all clones belonging to the specified ClusterID. The enlist command spools all CloneiDs that contain ClusterID. The following settings are used for inclusion of GCG (Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wisconsin 53704):

Word size 7 Minimum overlap 14 Stringency 0.8 Minimum compliance 14 Maximum spaces 10 Significance of gaps 8 Significance of length 2 Arrangement results GCG

based on sequence information in CloneiD. “Kontig is the alignment of DNA sequences based on regions of homology between these sequences. A new sequence was created based on the ordered sequences into the contig. A contig containing CloneiD was identified and ambiguous sites of the sequence were edited based on the CloneiD alignment (SEQ ID NO: 27 to SEQ ID NO: 32) to give the best possible sequence. The procedure was repeated for all six CloneiDs listed in Table 6. This produces five unique contigs. Edited sequences of 5 contigs with the program

Michigan consensus sequence. Contig 2511785 overlaps with contig 3506132 and this new contig is called 2535 (SEQ ID NO: 33). Sequencher contigs were copied in the Sequence Analysis software package GCG.

Sequencher (Gene Codes 48,105). The assemblies were transferred to Software Corporation, Ann Arbor,

Each contig was translated into all six reading frames in the protein sequences. The sequences of alp5 (ΜΑ29) and Δ6 (ΜΑ524) M. alpina were compared to each translated contig using a FastA survey (a similarity survey between the query sequence and a group of sequences of the same type (nucleic acid or protein) by Pearson and Lipman). Homology between these sequences indicates the open reading frames of each contig. Homologies between Δ5 and Δ6 M. alpina with contigs 2535 and 3854933 were used to create a final contig called 25358a. Figure 13 is the pairing of FastA final contig 253538a and MA29 and Figure 14 is the pairing of FastA final contig 25358a and MA524. The DNA sequences of the different contigs are present in SEQ ID NO: 2 to SEQ ID NO: 33. The various peptide sequences are shown in SEQ ID NO: 34 to SEQ ID NO: 40.

Although an open reading frame was created by joining two contigs, contig 2535 indicates that there is a single sequence at the beginning of the contig that does not pair with contig 3854933. Therefore, it is possible that these contigs originated from independent desaturase as human genes.

Kontig 253538a contains an open reading frame encoding 432 amino acids. It starts with Gin (CAG) and ends with a stop codon (TGA). Kontig 253538 associates with the M. alpina Δ5 and Δ6 sequences, indicating that it is either any desaturase or other known desaturases that share homology with each other. These individual contigs listed in Table 18 as well as the intermediate contig 2535 and the final contig 253538a can be used to isolate whole human desaturase genes.

Use of human desaturases

These human sequences can be expressed in yeast and in plants using the procedures described in the Examples. When expressed in mammalian cells and in transgenic animals, these genes can provide codon bias. These human sequences can also be used to identify related desaturase sequences.

Table 6

Desaturase section CloneID from database LifeSeq Key word 151-300 Δ5 3808675 Desaturase fatty acid 301-446 Δ5 354535 Δ6 151-300 Δ6 3448789 Δ6 151-300 Δ6 1362863 Δ6 151-300 Δ6 2394760 Δ6 301-457 Δ6 3350263 Δ6

Example 13:

I. Infant formulas

A. Iron-containing Isomil® Soy Nutrition

Use: Used as a drink for infants, children and adults with allergy or sensitivity to cow's milk. Food for patients with lactose-related disorders: lactase insufficiency, lactose intolerance and galactosemia. Features:

• Contains soy protein isolate to prevent symptoms of allergy or sensitivity to cow's milk protein.

• Nutrition does not contain lactose to prevent lactose-related diarrhea.

• Low osmolality (240 mOsm / kg water) reduces the risk of osmotic diarrhea.

• Dual carbohydrates (maize and sucrose) used to increase carbohydrate absorption and reduce the risk of exceeding the absorption capacity of damaged intestines.

»O o o v v v η η η η η» η »» »» »» »» »» »» »» 1.8 mg of iron (in the form of ferrous sulphate) per 100 calories helps prevent iron deficiency.

• Contains the recommended dose of vitamins and minerals.

• Contains vegetable oils that provide the recommended amount of essential fatty acids.

• The nutrition has a milky white color, a milk-like consistency and a pleasant aroma.

Ingredients: (Pareve,) 85% water, 4.9% corn syrup, 2.6% sugar (sucrose), 2.1% soy protein isolate, 1.4% coconut oil, 0.15% calcium citrate, 0, 11% tribasic calcium phosphate, potassium citrate, monobasic potassium phosphate, potassium chloride, mono- and diglycerides, soya lecithin, carrageenan, ascorbic acid, L-methionine, magnesium chloride, dibasic potassium phosphate, sodium chloride, choline chloride, taurine, iron sulphate, m-inositol, alpha-tocopheryl acetate, sulfate, zinc L-carnitine, niacinamide, calcium pantothenate, cupric sulfate, vitamin a palmitate, thiaminchloridhydrochlorid, riboflavin, pyridoxine hydrochloride, folic acid, potassium iodide, phylloquinone, biotin, sodium selenite, vitamin D ₃ and cyanocobalamin.

B. Isomyl® DF soya food, which is applied in case of diarrhea

Application: Suitable for short-term application in case of diarrhea in infants and toddlers.

Features:

• The first infant formula, which contains added soy fiber and which is applied specifically during diarrhea.

• Based on a clinical study, it has been shown to reduce the duration of mushy, watery stools during moderate and severe diarrhea in infants.

• Satisfies the nutritional needs of infants.

• Soy protein isolate with added L-methionine satisfies or exceeds the requirements of infants for all essential amino acids.

• The product does not contain lactose, thus preventing diarrhea associated with lactose.

• Low osmolality (240 mOsm / kg water) reduces the risk of osmotic diarrhea.

• Dual carbohydrates (corn syrup and sucrose), used to increase carbohydrate absorption and reduce the risk of exceeding the absorption capacity of damaged intestines.

• Meets or exceeds the amount of vitamins and minerals recommended by the Committee on Nutrition of the American Academy of Pediatrics.

• 1.8 mg of iron (in the form of ferrous sulphate) per 100 calories helps prevent iron deficiency.

• Contains vegetable oils that provide the recommended amount of essential fatty acids.

Ingredients: (Pareve,) 86% water, 4.8% corn syrup, 2.5% sugar (sucrose), 2.0% soy protein isolate, 2.1% soybean oil, 1.4% coconut oil, 0, 77% soya fiber, calcium, 0.11 i ϊ tribasic potassium citrate, monobasic phosphate

0.12% calcium, potassium, lecithin, citrate

0,10 potassium chloride, mono- and diglycerides, soya carrageenan, ascorbic acid, L-methionine, magnesium chloride, pyazic potassium phosphate, sodium chloride, choline chloride, taurine, ferric sulphate, m-inositol, alfatocopheryl acetate, zinc sulphate, L-carnitine , niacinamide, calcium pantothenate, copper sulfate, vitamin A palmitate, thiamine chloride hydrochloride, riboflavin, pyridoxine hydrochloride, folic acid, potassium iodide, phylloquinone, biotin, sodium selenite, vitamin D3 and cyanocobalamin.

C. Isomil® SF soya food with sucrose-free iron

Use: As a drink for infants, children and adults with allergy or sensitivity to cow's milk. Food for patients with lactose-related disorders: lactase insufficiency, lactose intolerance and sucrose.

Features:

• Contains soy protein isolate to prevent symptoms of allergy or sensitivity to cow's milk protein.

• Nutrition does not contain lactose to prevent lactose-related diarrhea (the source of carbohydrates is Polycose® Glucose Polymers).

• Sucrose-free, making it suitable for patients who do not tolerate sucrose.

• Low osmolality (180 mOsm / kg water) reduces the risk of osmotic diarrhea.

• 1.8 mg of iron (in the form of ferrous sulphate) per 100 calories helps prevent iron deficiency.

• Contains the recommended dose of vitamins and minerals.

• Contains vegetable oils that provide the recommended amount of essential fatty acids.

• The nutrition has a milky white color, a milk-like consistency and a pleasant aroma.

Ingredients: (Pareve) 75% water, 11.8% hydrolyzed corn starch, 4.1% soybean oil, 4.1% soy protein isolate, 2.8% coconut oil, 1.0% modified corn starch, 0.38 % tribasic calcium phosphate, 0.17% potassium citrate, 0.13% potassium chloride, mono- and diglycerides, soya lecithin, carrageenan, ascorbic acid, L-methionine, magnesium chloride, calcium carbonate, sodium chloride, choline chloride, taurine, sulfate iron (III), m-inositol, alpha-tocopheryl acetate, zinc sulfate, L-carnitine, niacinamide, calcium pantothenate, cupric sulfate, vitamin A palmitate, thiamine chloride hydrochloride, riboflavin, pyridoxine hydrochloride, folic acid, magnesium sulfate, potassium iodide, sodium selenium, biotinquinone, , vitamin D ₃ and cyanocobalamin.

«·

D. Soy nutrition Isomyl®20 with iron (for direct consumption) 20 calories per 0,0284 1

Use: if soy nutrition is required

Ingredients: (Pareve,) 85% water, 4.9% corn syrup, 2.6% sugar (sucrose), protein isolate, calcium, 0.11%

2.1% soybean oil, 1.9% soybean oil

1,4% coconut oil, 0,15% tribasic calcium phosphate citrate, potassium citrate, monobasic potassium phosphate, potassium chloride, mono- and diglycerides, soya lecithin, carrageenan, ascorbic acid, L-methionine, magnesium chloride, dibasic potassium phosphate, sodium chloride, choline chloride, taurine, ferric sulfate, m-inositol, alpha-tocopheryl acetate, zinc sulfate, L-carnitine, niacinamide, calcium pantothenate, copper sulfate, vitamin A palmitate, thiamine chloride hydrochloride, riboflavin, pyridoxine hydrochloride, folate, magnesium sulfate, potassium, phylloquinone, biotin, sodium selenite, vitamin D ₃ and cyanocobalamin.

E. Similac® Baby Food

Use: if an infant formula is needed: if it is necessary to discontinue breast-feeding before reaching one year of the baby, if it is necessary to supplement breast-feeding. Can be used as feed if breast feeding is not possible.

Features:

• Contains protein of suitable quality and quantity to ensure good growth. The protein is denatured by heat, reducing the risk of rectal bleeding associated with milk administration.

• Contains fat from a blend of vegetable oils (twice homogenized), providing linolenic acid that is easily absorbable.

• Contains carbohydrates such as lactose in a ratio that is similar to human milk.

• Low in renal solutes, minimizing stress on developing organs.

• It is available in powder form, concentrated solution and ready to use form.

Ingredients: (-D) water, non-fat milk, lactose, soybean oil, coconut oil, mono- and diglycerides, soybean lecithin, carrageenan, ascorbic acid, choline chloride, taurine, m-inositol, alfatocopheryl acetate, zinc sulfate, niacinamide, ferrous sulfate, calcium pantothenate, copper sulfate, vitamin A palmitate, thiamin chloride hydrochloride, riboflavin, pyridoxine hydrochloride, folic acid, magnesium sulfate, phylloquinone, biotin, sodium selenite, vitamin D ₃ and cyanocobalamin.

F. Infant formulas for preterm infants with Similac®NeoCare iron

Use: Suitable for premature infants requiring special nutrition after discharge from hospital. Similac NeoCare is a nutritionally complete nutrition developed for premature infants that contains the extra calories, protein, vitamins and minerals needed to induce growth and support development.

Features:

• Reduces the need for caloric and vitamin supplements. Compared to the standard nutrition described (20 calories per 0.0284 l), it contains more calories (22 calories per 0.0284 l).

• Contains a highly absorbable blend of medium-chain triglycerides (MCT oil) to help meet the special digestive needs of premature infants.

• It contains higher amounts of protein, vitamins and minerals per 100 calories to increase the initial dose of nutrients given in the hospital.

• Contains more calcium and phosphorus, allowing bone mineralization.

Ingredients: -D corn syrup solids, nonfat milk, lactose, whey protein concentrate, soybean oil, sunflower oil, fractionated coconut oil (medium chain triglycerides), potassium citrate, tribasic calcium phosphate, calcium carbonate, ascorbic acid, magnesium chloride , potassium chloride, sodium chloride, taurine, ferric sulphate, m-inositol, alpha-tocopheryl acetate, zinc sulphate, L-carnitine, niacinamide, calcium pantothenate, copper sulphate, vitamin A palmitate, thiamine chloride hydrochloride, riboflavin, beta-carotene, pyridoxine hydrochloride, folic, magnesium sulfate, phylloquinone, biotin, sodium selenite, vitamin D3 and cyanocobalamin.

G. Low Milk Human Milk Enriched With Similac Natural Care In Ready For Use, 24 Cal. to 0.0284 1

Use: The product is intended for mixing with human milk or for use as an alternative to human milk in infants with low birth weight.

Ingredients: -D water, non-fat milk, hydrolyzed corn starch, lactose, whey protein concentrate, soybean oil, sunflower oil, fractionated coconut oil (medium chain triglycerides), coconut oil, potassium citrate, tribasic calcium phosphate, calcium carbonate, mono- and diglycerides, soya lecithin, carrageenan, choline chloride, taurine, m-inositol, alpha-tocopheryl acetate, zinc sulfate, L-carnitine, potassium chloride, calcium pantothenate, copper sulfate, vitamin A palmitate, thiamine chloride hydrochloride, pyridoxine hydrochloride, folic acid, magnesium sulfate, phylloquinone, biotin, sodium selenite, vitamin D3 and cyanocobalamin.

• ·

The PUFAs according to the invention may be added to these nutrients or they may replace some of the constituents of said nutrients.

II. Nutrition

A. ENSURE®

Usage: ENSURE is a liquid nutrition containing small amounts of residues prepared primarily as an oral nutritional supplement to be taken with or between meals or in appropriate amounts as a meal substitute. ENSURE does not contain lactose and gluten and is suitable for use on modified diets such as low cholesterol diets. Although primarily administered as an oral supplement, it can be applied by tubing.

Patient Status:

• Patients on a modified diet • Patients with old age with specific dietary requirements • Patients who involuntarily lose weight • Patients after illness or surgery • Patients in need of a low-residue diet.

Ingredients: -D water, sugar (sucrose), maltodextrin (maize), calcium and sodium caseinate, sunflower oil, soy protein isolate, soybean oil, canola oil, potassium, tribasic calcium phosphate, sodium citrate, artificial dibasic magnesium phosphate, chloride magnesium, additives, sodium chloride, soya lecithin, choline chloride, ascorbic acid, carrageenan, zinc sulfate, ferrous sulfate, alpha-tocopheryl acetate, gelatin gum, niacinamide, calcium pantothenate, magnesium sulfate, copper sulfate, vitamin A palmitate, thiamine chloride hydrochloride, pyridoxine hydrochloride, pyridoxine , folic acid, sodium molybdate, chromium chloride, biotin, potassium iodide, sodium selenate.

B. ENSURE®BARS

Use: ENSURE BARS is a total, balanced nutrition that is used as a supplement between meals or is served with food. It is a delicious, nutritionally rich alternative to other desserts. ENSURE BAR contains less than 1 g lactose per bar (Chocolate Fudge Brownie does not contain gluten, Honey Graham Crunch does contain gluten).

Patient Status:

• Patients who need more calories, proteins, vitamins and minerals.

• Particularly suitable for people who do not receive enough calories and nutrients.

• Suitable for people who are able to chew and swallow • Not suitable for people allergic to peanuts and any allergy to nuts.

Folders:

Honey Graham Crunch type contains high fructose corn syrup, soy protein isolate, brown sugar, maltodextrin (corn), crispy rice (crushed rice, sugar (sucrose), salt (sodium chloride) and malt), oat bran, partially hydrogenated oil from cotton seeds and soybean oil, soy polysaccharides, glycerin, whey protein concentrate, polydextrose, fructose, calcium caseinate, cocoa powder, artificial flavors, canola oil, sunflower oil, nonfat milk powder, whey powder, soy lecithin, corn oil. These bars are made in nuts processing plants.

Vitamins and minerals:

Tribasic calcium phosphate, dibasic potassium phosphate, magnesium oxide, salt (sodium chloride), potassium chloride, ascorbic acid, ferric phosphate, alphatocopheryl acetate, niacinamide, zinc oxide, pantothenate

calcium, copper gluconate, manganese sulfate, riboflavin, betacarotene, pyridoxine hydrochloride, thiamine nitrate, folic acid, biotin, chromium chloride, potassium iodide, sodium selenate, sodium molybdate, phylloquinone, vitamin D3, and cyanocobalamin.

Protein:

Honey Graham Crunch type: Source of protein isolate proteins and milk proteins. Soy protein isolate

Milk proteins

Fat:

A: The source of fat is a mixture of partially and seeds of cotton, sunflower and lecithin.

soybean and cotton seed76%

Honey Graham Crunch type of hydrogenated soybean oil and soybean oil Partially hydrogenated sunflower oil canola oil soybean lecithin is a mixture of soybean%

%

Carbohydrates: Honey Graham Crunch type: Carbohydrate source is a combination corn syrup with high fructose content, Brown sugar, maltodextrin, honey, crispy rice, glycerin, soy polysaccharides and oat bran. corn syrup with high fructose content 24% Brown sugar 21% maltodextrin 12% copper 11% crispy rice 9% glycerine 9% soy polysaccharide 7% Oat bran 7%

C. ENSURE® HIGH PROTEIN

Use: ENSURE® HIGH PROTEIN is a concentrated, high-protein liquid nutrition for people who need extra calories, protein, vitamins and minerals. It can be administered orally, as a nutritional supplement between meals, or given with food. In some quantities it can replace food. ENSURE® HIGH PROTEIN is lactose-free and gluten-free and is suitable for application to people after surgery, neck fractures and patients who have a risk of ulcers.

Patient Status:

• The product is suitable for a patient in need of a higher intake of calories, protein, vitamins, minerals such as patients after surgery or cervical fracture, patients suffering from pressure ulcers and patients on a low-cholesterol diet.

Features:

• Low saturated fat content • Contains 6 g total fat and less than 5 mg cholesterol per package • Rich and mild taste • Great source of protein, calcium and other essential vitamins and minerals • Suitable for low cholesterol diet • Lactose-free, easy is being spent.

Folders:

Vanilla Supreme Type: - -D water, sugar (sucrose), maltodextrin (maize), calcium and sodium caseinate, sunflower oil, soy protein isolate, soybean oil, canola oil, potassium citrate, tribasic calcium phosphate, sodium citrate, magnesium chloride, dibasic magnesium phosphate, artificial

• · ««

• ingredients, sodium chloride, soya lecithin, choline chloride, ascorbic acid, carrageenan, zinc sulfate, ferrous sulfate, alpha-tocopheryl acetate, gelatin gum, niacinamide, calcium pantothenate, manganous sulfate, copper sulfate, vitamin A palmitate, thiamine chloride hydrochloride, pyridoxine hydrochloride, pyridoxine hydrochloride , folic acid, sodium molybdate, chromium chloride, biotin, potassium iodide, sodium selenate, phylloquinone, vitamin D3 and cyanocobalamin.

Protein:

The protein source is a mixture of two highly biologically valuable proteins: casein and soy.

Sodium and calcium caseinate 85%

Soy protein isolate 15%

Fat:

The source of fat is a mixture of three oils: sunflower oil, canola oil and soybean oil.

Sunflower oil 40%

Canola oil 30%

Soybean oil 30%

The amount of fat in ENSURE® HIGH PROTEIN meets the requirements of the American Heart Association (AHA). 6 grams of fat in ENSURE® HIGH PROTEIN represents 24% of total calories, with 2.6% of the fat coming from saturated fatty acids and 7.9% from polyunsaturated fatty acids. These values meet AHA requirements, which means less than 30% calories come from fat, less than 10% calories come from saturated fatty acids and less than 10% calories come from polyunsaturated fatty acids.

Carbohydrates:

ENSURE® HIGH PROTEIN contains a combination of maltodextrine and sucrose. Medium sweetness and a variety of flavors (vanilla, chocolate, wild berries and banana) plus VARI-FLAVORSO® Flavor Pacs in pecan, sour cherry, strawberry, lemon and orange nut, helps prevent bad taste.

Vanilla and other non-chocolate flavors include:

Sucrose 60%

Maltodextrin 40%

Chocolate flavor contains:

Sucrose 70%

Maltodextrin 30%

D. ENSURE®LIGHT

Usage: ENSURE LIGHT is a liquid nutrition containing a small amount of fat. It is used as an oral nutritional supplement administered between meals or with meals. ENSURE LIGHT is lactose-free and gluten-free and is suitable for use on a modified diet that includes low-cholesterol diets.

Patient status:

• Suitable for normal weight and overweight patients who need extra nutrients. It is given as a supplement that contains 50% less fat and 20% less calories than ENSURE.

• For healthy adults who do not eat properly and need extra nutrition.

Features:

• Contains small amounts of fat and saturated fat.

• Contains 3 g total fat and less than 5 mg cholesterol • The product has a rich mild taste • • • It is a great source of calcium and other essential vitamins and minerals • The product is suitable for a low cholesterol diet • The product is lactose-free and easy to digest .

Ingredients:

French Vanilla type: -D water, sugar (sucrose), maltodextrin (maize), calcium and sodium caseinate, sunflower oil, soy protein isolate, soybean oil, canola oil, magnesium chloride, potassium citrate, tribasic calcium phosphate, sodium citrate, magnesium chloride, dibasic magnesium phosphate, natural and artificial additives, cellulose, gel, choline chloride, soya lecithin, sodium chloride, ascorbic acid, carrageenan, cellulose gum, ferrous sulphate, alphatocopheryl acetate, zinc sulphate, niacinamide, calcium pantothenate, manganese sulphate, , vitamin A palmitate, thiaminochioride hydrochloride, pyridoxine hydrochloride, riboflavin, folic acid, sodium molybdate, chromium chloride, biotin, potassium iodide, sodium selenate, phylloquinone, vitamin D3 and cyanocobalamin.

Protein:

The protein source is calcium caseinate. Calcium caseinate

100 ALIGN!

Fat:

The protein source is a mixture of two oils: sunflower oil and canola oil.

Sunflower oil 70%

Canola oil 30%

The amount of fat in ENSURE LIGHT meets the requirements

American Heart Association (AHA). 3 grams of fat in the preparation

ENSURE LIGHT represents 13.5% of total calories, while 1.4% of the fat is from saturated fatty acids 2.6% of poly-unsaturated fatty acids. These values are in accordance with AHA requirements, with less than 30% calories from fat and less than 10% calories from saturated fatty acids and less than 10% of total calories from polyunsaturated fatty acids.

Carbohydrates:

ENSURE LIGHT contains a combination of maltodextrine and sucrose. The chocolate flavor also contains corn syrup. Medium sweetness and a variety of flavors (vanilla, chocolate, strawberry) plus VARI-FLAVORSO® Flavor Pacs in pecan, sour cherry, strawberry, lemon and orange, helps prevent bad taste.

Vanilla and other non-chocolate flavors include:

Sucrose 51%

Maltodextrin 49%

Chocolate flavor contains:

Sucrose 47%

Corn syrup 26,5%

Maltodextrin 26,5%

Vitamins and minerals

One package (0.2272 1) ENSURE LIGHT provides at least 25%

RDI for 24 key vitamins and minerals.

Caffeine:

The chocolate flavor contains 2.1 mg of caffeine per 0.2272 L.

E. ENSURE PLUS®

Usage: ENSURE PLUS is a high calorie nutrition in liquid form with a low content of residues. It is used where extra calories and nutrients are needed but normal

0)? ····

-7 4-. Protein concentration. The preparation) serves primarily as an oral nutritional supplement to be used between an individual meal or in an acceptable amount as a meal substitute. ENSURE PLUS does not contain lactose or gluten. Although the product is primarily administered orally, it can also be passed through the tubing.

Patient Status:

• The product is suitable for patients who require extra calories and nutrients in a limited volume but normal protein concentration. • The product is suitable for patients who need to achieve and maintain normal weight.

Features:

• Rich and delicious taste • Good source of essential vitamins and minerals

Ingredients:

Vanilla type: -D water, corn syrup, maltodextrin (corn), sugar (sucrose), corn oil, calcium and sodium caseinate, sunflower oil, soy protein isolate, magnesium chloride, potassium citrate, tribasic calcium phosphate, soy lecithin, natural and artificial flavors, sodium citrate, potassium chloride, choline chloride, ascorbic acid, carrageenan, zinc sulfate, copper sulfate, ferric sulfate, alphatocopheryl acetate, zinc sulfate, niacinamide, calcium pantothenate, manganese sulfate, copper sulfate, vitamin A palmitate, thiamine chloride hydrochloride, riboflavin, folic acid, sodium molybdate, chromium chloride, potassium iodide, sodium selenate, phylloquinone, vitamin D3, and cyanocobalamin.

Protein:

• ·

The protein source is two highly biologically valuable proteins: casein and soy

Calcium and sodium caseinate 84%

Soy protein isolate 16%

Fat:

The source of fat is corn oil

Corn oil 100%

Carbohydrates:

ENSURE PLUS contains a combination of maltodextrin and sucrose. Medium sweetness and a variety of flavors (vanilla, chocolate, strawberry-coffee, peanut butter and egg cognac) plus VARI-FLAVORSO® Flavor Pacs in pecan, sour cherry, strawberry, lemon and orange help prevent bad taste.

Vanilla, strawberry, coffee nuts contain:

Sucrose Maltodextrin Corn syrup

Chocolate flavor and sucrose Corn syrup Maltodextrin peanut butter flavor%

%%

Cognac contains:

%%

%

Vitamins and minerals

One package (0.2272 1) ENSURE LIGHT provides at least 15%

RDI for 25 key vitamins and minerals.

Caffeine:

The chocolate flavor contains 3.1 mg caffeine per 0.2272 L and the coffee flavor contains trace amounts of caffeine.

• *

F. ENSURE PLUS®HN

Usage: ENSURE PLUS HN is a nutritionally complete nutrition in liquid form with a high calorie and nitrogen content suitable for people who require a large amount of calories and protein and have limited food tolerance. They can be administered orally or through a tube. The product does not contain lactose or gluten.

Patient Status:

• Suitable for patients with an increased need for calories and proteins, such as after surgery or injury.

• Suitable for patients with limited volume tolerance and early satiety.

Features:

• The product is suitable both as a nutritional supplement and as a complete nutrition.

• The product is suitable for oral administration as well as tubing.

• 1.5 CaVml • High nitrogen content.

• High calorie density.

Folders:

Vanilla type: -D water, maltodextrin (maize), corn oil, calcium and sodium caseinate, sugar (sucrose), soy protein isolate, magnesium chloride, potassium citrate, tribasic calcium phosphate, soy lecithin, natural and artificial flavors, sodium citrate , choline chloride, ascorbic acid, taurine, L-carnitine, alpha-tocopheryl acetate, zinc sulphate, niacinamide sulphate, carrageenan, ferric, calcium pantothenate, manganese sulphate, copper sulphate, * · φ φ φ * φ φ * * * * * * y y y i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i vitamin A palmitate, folic acid, biotin, sodium molybdate, chromium chloride, potassium iodide, sodium selenate, phylloquinone, vitamin D3, and cyanocobalamin.

G. ENSURE® POWDER

Usage: ENSURE POWDER (dissolved in water) is a low-residue liquid nutrient intended for application primarily as an oral nutritional supplement to be used with or with individual meals. The composition is lactose-free and gluten-free and is suitable for modified diets including low-cholesterol diets.

Patient Status:

• The product is suitable for patients on a special diet.

• The product is suitable for the elderly where there is a risk of nutrition.

• The product is suitable for patients who are recovering from illness or surgery.

• The product is suitable for patients who need a low-residue diet.

Features:

• The product is pleasant, easy to mix.

• Contains a small amount of saturated fat.

• Each pack contains 9g of total fat and less than 5mg of cholesterol.

• The product contains a high content of vitamins and minerals.

• The product is suitable for a low cholesterol diet.

• The product does not contain lactose and is easy to digest.

Ingredients: - -D maize syrup, maltodextrin (maize), corn oil, calcium and sodium caseinate, sugar (sucrose), «·> • · * 9999 soy protein isolate, artificial flavors, magnesium chloride, potassium citrate, tribasic calcium phosphate, potassium chloride, soya lecithin, sodium citrate, choline chloride, ascorbic acid, zinc sulfate, ferric sulfate, taurine, L-carnitine, alpha-tocopheryl acetate, niacinamide, calcium pantothenate, manganese sulfate, copper sulfate, thiamine chloride hydrochloride, pyridoxine hydrochloride, riboflavin hydrochloride, , folic acid, biotin, sodium molybdate, chromium chloride, potassium iodide, sodium selenate, phylloquinone, vitamin D3, and cyanocobalamin.

Protein:

A protein source is a mixture of two highly biologically valuable proteins: casein and soy.

Sodium and calcium caseinate 84%

Soy protein isolate 16%

Fat:

The source of fat is corn oil.

Corn oil 100%

Carbohydrates:

ENSURE POWDER contains a combination of corn syrup, maltodextrin and sucrose. Medium sweetness plus VARIFLAVORSO® Flavor Pacs in pecan, sour cherries, strawberries, lemon and orange, helps prevent bad taste.

Vanilla flavor contains:

Sucrose 30%

Maltodextrin 35%

Corn syrup 35%

H. ENSURE® PUDDING · · · · · · · · · · · · · · · · · ·

Use: ENSURE PUDDING is a nutritional supplement with a high density of nutrients providing balanced nutrition in a non-liquid form. It is used between and with meals. It is suitable for nutrition with a modified consistency (for example, a fine, comminuted or completely liquid diet) or for people with swallowing disorders. Said preparation does not contain gluten.

Patient Status:

• The product is suitable for patients on a consistency-adjusted diet (for example, a soft, comminuted or completely liquid diet).

• Suitable for patients with swallowing disorders.

Features:

• Rich and soft, good taste.

• A good source of essential vitamins and minerals. It must not be stored in refrigerators.

• Gluten-free.

Nutritional profile for volume 0,142 ml: 250 calories, 10.9% proteins, total fat content is 34.9%, 54.2 % carbohydrate. Ingredients: : - -D non-fat milk, water, sugar (sucrose), partly hydrogenated soybean oil, modified food

starch, magnesium sulfate, sodium stearoyl lactylate, dibasic sodium phosphate, artificial flavors, ascorbic acid, zinc sulfate, iron sulfate, alpha-tocopheryl acetate, choline chloride, niacinamide, manganese sulfate, calcium pantothenate, potassium citrate, copper sulfate, vitamin A palmitate, thiamine chloride, pyridoxine hydrochloride, riboflavin, FD&C yellow # 6, folic acid, biotin, phylloquinone, vitamin D3 and cyanocobalamin.

· · · · · · ·

Protein:

The protein source is nonfat milk.

Non-fat milk 100%

Fat:

The source of fat is hydrogenated soybean oil. Hydrogenated soybean oil 100%

Carbohydrates:

ENSURE PUDDING contains a combination of sucrose and modified food starch. Medium sweetness and a variety of flavors (vanilla, chocolate, scotch and sago helps prevent bad taste. The product contains 9.2 grams of lactose per pack.

Vanilla and other non-chocolate Sucrose

Lactose

Prepared food starch

Chocolate flavor

Sucrose

Lactose

The modified food starch flavors include:

%%

%%

%%

I. ENSURE® WITH FIBER

Use: ENSURE® WITH FIBER is a nutritionally complete nutrition in liquid form, containing fiber intended for people who prefer to apply a diet with a higher fiber and nutrient content. The product is suitable for people who do not require a low residue diet. It can be applied orally or through a tube. The product may be used as a nutritional supplement to a normal diet or in a suitable amount to replace it. The product does not contain lactose or gluten and is suitable for use in modified diets, including a low cholesterol diet.

Patient Status:

• A patient for whom a diet with a higher fiber and nutrient content is beneficial.

Features:

• New progressive product with low saturated fat content, higher vitamins and minerals • Contains 6 g of total fat and less than 5 mg of cholesterol in one package.

• It has a rich and delicate taste.

• Good fiber source.

• Excellent source of essential vitamins and minerals.

• Suitable for a low cholesterol diet.

• Lactose and gluten free.

Ingredients:

Vanilla type: -D water, maltodextrin (maize), sugar (sucrose), corn oil, calcium and sodium caseinate, oat fiber, sunflower oil, canola oil, soy protein isolate, corn oil, soy fiber, tribasic calcium phosphate, chloride magnesium, potassium citrate, potassium citrate, cellulose gel, dibasic potassium phosphate, sodium citrate, natural and artificial flavors, choline chloride, magnesium phosphate, ascorbic acid, cellulose gum, potassium chloride, carrageenan, ferric sulfate, alpha-tocopheryl acetate, calcium pantothenate, sulfate manganese, copper sulfate, vitamin A palmitate, thiamine chloride hydrochloride, pyridoxine hydrochloride, riboflavin, folic acid, biotin, sodium molybdate, chromium chloride, iodide • ·

100 potassium, sodium selenate, phylloquinone, vitamin D3 and cyanocobalamin.

Protein:

The protein source is a mixture of two highly biologically valuable proteins: caseins and soy.

Sodium and calcium caseinate Soy protein isolate 80% 20% Fat: The source of fat is a mixture of three oils: sunflower oil, canola oil and corn oil. Sunflower oil 40% Oil canola 40% Corn oil 20% The amount of fat in ENSURE WITH FIBER meets American Heart Association (AHA) requirements. 6 grams of fat of that product represents 22% of the total quantity

calories, 2.01% is fat derived from saturated fatty acids and 6.7% comes from polyunsaturated fatty acids. These values are in accordance with AHA requirements where less than 30% of the total calories come from fat, less than 10% of the calories come from saturated fatty acids and less than 10% from the total calories come from polyunsaturated fatty acids.

Carbohydrates:

ENSURE WITH FIBER contains a combination of maltodextrine and sucrose. Medium sweetness and a variety of flavors (vanilla, chocolate and pecan butter) plus VARI-FLAVORSO® Flavor Pacs in pecan, sour cherry, strawberry, lemon and orange nut, helps prevent bad taste.

Vanilla and other non-chocolate flavors • ·

101

Sucrose

Maltodextrin

Oat fiber Soya fiber%

%%

%

Chocolate flavor

Sucrose 36%

Maltodextrin 55%

Oat fiber 7%

Soya fiber 2%

Fiber:

The fiber blend contained in ENSURE WITH FIBER contains oat fiber and soy polysaccharide. As a result of this mixture, a single 0.22721 package contains approximately 4 grams of total fiber. The ratio of insoluble to soluble fiber is 95: 5.

The PUFAs of the invention may replace and / or supplement the various nutritional supplements described above.

J. Oxepa ™ nutritional product

Oxepa is a low calorie enteral nutritional product with a high calorie density suitable for administration to patients suffering from or at risk of ARDS. The product has a unique combination of ingredients including a patented oil blend containing icosapentanoic acid (fish oil EPA), γ-linolenic acid (GLA derived from borage oil) and an increased amount of antioxidants.

Calorie breakdown:

• Caloric density is high at 1.5 calories / ml (355 calories / 8 fl.oz.), minimizing the volume required to meet energy requirements.

• The distribution of calories in Oxepa is shown in Table no.

.

Table 7: Calorie distribution in Oxepa

to 8 f1. oz. per liter % cal. Calorie 355 1 500 - Fat (g) 22.2 93.7 55.2 Carbohydrates (g) 25 105.5 28.1 Protein (g) 14.8 62.5 16, 7 Water (g) 186 785 -

Fat:

• Oxepa contains 22.2 grams of fat per fl. oz. (93.7 g / L).

• The fat source is a blend of oils containing 31.8% canola oil, 25% medium chain triglycerides (MCT), 20% borage oil, 20% fish oil and 3.2% soya lecithin. The typical fatty acid profile of Oxepa is shown in Table 8.

• Oxepa provides a balanced amount of polyunsaturated, monounsaturated and saturated fatty acids, as shown in Table 10.

• Medium chain triglycerides (MTCs), which form a% of the mixture, allow the gastric tract to be emptied as they are absorbed in the intestinal tract without being emulsified with bile acid.

The PUFAs of the invention may replace or supplement the various fatty acid components of the Oxepa nutritional product.

Table 8: Typical fatty acid profile

% of total fatty acids g / 8 fl. oz. * g / 1 * Acid 0.2 0.04 0.18

103

Kapron (6: 0) Caprylic acid (8: 0) 14, 69 3.1 13.07 Capric acid (10: 0) 11.06 2.33 9, 87 Acid palmitová (16: 1) 5.59 1, 18 4, 98 Palmitoleic acid (16: ln-7) 1, 82 0.38 1, 62 Stearic acid (18: 0) 1, 84 0.39 1, 64 Oleic acid (18: ln-9) 24.44 5.16 21.75 Acid linoleum 16.28 3.44 14.49 Acid α-linol (18: 3n-3) 3, 47 0.73 3.09 Γ-Linoleic acid (18: 3n-6) 4, 82 1,02 4.29 Icosapentanoic acid (20: 5n-3) 5.11 1.08 4.55 n-3- docosapentane (22: 5n-3) 0.55 0.12 0.49 Docosahexanoic acid (22: 6n-3) 2.27 0.48 2.02 Other acids 7.55 1.52 6.72

* fatty acids are approximately 95% of total fat

Table 9: Fat profile of Oxepa

% of total calories from fat 55.2 Polyunsaturated fatty acids 31.44 g / l Monounsaturated fatty acids 25.53 g / l Saturated fatty acids 32.38 g / l The ratio of n-6 to n-3 1.75: 1 cholesterol 9.49 mg / 8 fl.oz. 40.1 mg / l

• ·

104 ··: ··;

··· ·· ······· ·· · ·

Carbohydrates:

• Carbohydrate content is 25.0 g per 8 fl.oz. (105.5 g / L).

• Sources of carbohydrates are 45% maltodextrin (complex carbohydrate) and 55% sucrose (simple sugar). Both carbohydrates are simply digested and absorbed.

• Oxepa contains high fat and low carbohydrate content to minimize carbon dioxide (CO2) production. Low carbohydrate content is also suitable for patients who may experience stress-induced hyperglycaemia.

• Oxepa does not contain lactose.

Carbohydrates, protein amino acids, and glycerol moieties can be converted to glucose in the body. Through this process, glucose-dependent tissue sugars (such as the central nervous system or blood cells) are satisfied. However, carbohydrate-free food can cause ketosis, which is excessive catabolism of tissue proteins and loss of body fluids and electrolytes. These effects can be prevented if 50 to 100 g of digestible carbohydrates are consumed daily if caloric intake is adequate. The amount of carbohydrates in Oxepa is also sufficient to minimize gluconeogenesis when energy needs are met.

Protein:

• Oxepa contains 14.8 g protein per package of 8 f1. oz. (62.5 g / l).

• The total calorie / nitrogen ratio (150: 1) meets the needs of stressed patients.

• Oxepa provides enough protein to cause anabolism and maintain low body weight without respiratory problems. High protein intake is expected in patients

105 with respiratory insufficiency. Although proteins have little effect on CO 2 production, a high protein diet aids in breathing.

• 86.8% sodium caseinate and 13.2% calcium caseinate are used as a protein source in Oxepa.

• As shown in Table 11, the amino acid profile of the protein system in Oxepa meets high quality protein standards developed by the National Academy of Sciences.

• Oxepa does not contain gluten.

• ·

106

Sequence Protocol (2) Information about SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 1617 pairs of cows (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: other nucleic acid XI) Description SEQ ID NO: 1:

CGACACTCCT TCCTTCTTCT CACCCGTCCT AGTCCCCTTC AACCCCCCTC TTTGACAAAG 60 ACAACAAACC ATGGCTGCTG CTCCCAGTGT GAGGACGTTT ACTCGGGCCG AGGTTTTGAA 120 TGCCGAGGCT CTGAATGAGG GCAAGAAGGA TGCCGAGGCA CCCTTCTTGA TGATCATCGA 180 CAACAAGGTG TACGATGTCC GCGAGTTCGT CCCTGATCAT CCCGGTGGAA GTGTGATTCT 240 CACGCACGTT GGCAAGGACG GCACTGACGT CTTTGACACT TTTCACCCCG AGGCTGCTTG 300 GGAGACTCTT GCCAACTTTT ACGTTGGTGA TATTGACGAG AGCGACCGCG ATATCAAGAA 360 TGATGACTTT GCGGCCGAGG TCCGCAAGCT GCGTACCTTG TTCCAGTCTC TTGGTTACTA 420 CGATTCTTCC AAGGCATACT ACGCCTTCAA GGTCTCGTTC AACCTCTGCA TCTGGGGTTT 480 GTCGACGGTC ATTGTGGCCA AGTGGGGCCA GACCTCGACC CTCGCCAACG TGCTCTCGGC 540 TGCGCTTTTG GGTCTGTTCT GGCAGCAGTG CGGATGGTTG GCTCACGACT TTTTGCATCA 600 CCAGGTCTTC CAGGACCGTT TCTGGGGTGA TCTTTTCGGC GCCTTCTTGG GAGGTGTCTG 660 CCAGGGCTTC TCGTCCTCGT GGTGGAAGGA CAAGCACAAC ACTCACCACG CCGCCCCCAA 720 CGTCCACGGC GAGGATCCCG ACATTGACAC CCACCCTCTG TTGACCTGGA GTGAGCATGC 780 GTTGGAGATG TTCTCGGATG TCCCAGATGA GGAGCTGACC CGCATGTGGT CGCGTTTCAT 840 GGTCCTGAAC CAGACCTGGT TTTACTTCCC CATTCTCTCG TTTGCCCGTC TCTCCTGGTG 900 CCTCCAGTCC ATTCTCTTTG TGCTGCCTAA CGGTCAGGCC CACAAGCCCT CGGGCGCGCG 960 TGTGCCCATC TCGTTGGTCG AGCAGCTGTC GCTTGCGATG CACTGGACCT GGTACCTCGC 1020 CACCATGTTC CTGTTCATCA AGGATCCCGT CAACATGCTG GTGTACTTTT TGGTGTCGCA 1080 GGCGGTGTGC GGAAACTTGT TGGCGATCGT GTTCTCGCTC AACCACAACG GTATGCCTGT 1140 GATCTCGAAG GAGGAGGCGG TCGATATGGA TTTCTTCACG AAGCAGATCA TCACGGGTCG 1200 TGATGTCCAC CCGGGTCTAT TTGCCAACTG GTTCACGGGT GGATTGAACT ATCAGATCGA 1260 GCACCACTTG TTCCCTTCGA TGCCTCGCCA CAACTTTTCA AAGATCCAGC CTGCTGTCGA 1320

• ·

107 «· • • • • · · • «· · · · • · · · • · · · · • · * · • · ······· • • • · · • · GACCCTGTGC AAAAAGTACA ATGTCCGATA CCACACCACC GGTATGATCG AGGGAACTGC 1380 AGAGGTCTTT AGCCGTCTGA ACGAGGTCTC CAAGGCTGCC TCCAAGATGG GTAAGGCGCA 1440 GTAAAAAAAA AAACAAGGAC GTTTTTTTTC GCCAGTGCCT GTGCCTGTGC CTGCTTCCCT 1500 TGTCAAGTCG AGCGTTTCTG GAAAGGATCG TTCAGTGCAG TATCATCATT CTCCTTTTAC 1560 CCCCCGCTCA TATCTCATTC ATTTCTCTTA TTAAACAACT TGTTCCCCCC TTCACCG 1617

(2) SEQ ID NO: 2 Information:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 457 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: peptide

(xi) Description sequence: SEQ ID NO: 2: Met 1 Ala Ala Ala Pro 5 Ser Val Arg Thr Phe Thr Arg 10 Ala Glu Val Leu Asn Ala Glu Ala Leu Asn Glu Gly Lys Lys Asp Ala Glu Ala Pro Phe Leu Met Ile Ile Asp Asn Lys Val Tyr Asp Val Arg Glu Phe Val Pro 45 Asp His Pro 50 Gly Gly Ser Val Ile Leu Thr His Val Gly Lys Thr 65 Asp Val Phe Asp Thr Phe His For Glu Ala Ala 75 Trp Glu Thr Leu Ala Asn Phe Tyr Val Gly Asp Ile Asp Glu Ser Asp 90 Arg Asp Ile Lys Asn Asp Asp Phe Ala Ala Glu Val Arg Lys Leu Arg Thr Leu Phe Gin 110 Ser Leu Gly Tyr Tyr Asp Ser Ser 120 Lys Ala Tyr Tyr Ala Phe Lys Val 125 Ser Phe Asn Leu Cys Ile Trp Gly Leu Ser Thr Val 140 Ile Val Ala Lys Trp 145 Gly Gin Thr Ser. Thr Leu Ala (150 Asn Val Leu Ser 155 Ala Ala Leu Leu

Gly Leu Phe Trp द्वारा किया Gin Gin Cys Gly Leu Ala His Asp

• · ·

His Gin Val

Leu Gly Gly 195

His Asn Thr

Ile Asp Thr 225

Phe Ser Asp

Met Leu

Arg Leu Ser 275

Gin Ala His 290

Gin Leu Ser 305

Leu Phe Ile

Gin Ala Val

Asn Gly Met 355

Phe Thr Lys

Ala Asn Trp 385

Phe Pro Ser

Glu Thr Leu

Ile Glu Gly 434

Ala Ala Ser 450

108

Phe Gin Asp Phe Gly Asp Leu Phe Gly Ala Phe 180 185 190

Val Cys Gin Phly Ser Ser Ser Trp Lys Asp Lys 200 205

His His Ala Ala Asn Val His Gly Glu Asp Pro Asp 215 220

His Leu Leu Thr Trp Ser Glu

Val Pro Asp Glu Glu Le Thr Arg Met Trp Ser Arg Phe 245 250 255

Asn Gin Thr Trp Phe Tyr Phe Pro Ile Leu Ser Phe Ala 260 265 270

Trp Cys Leu Phe Val Leu For Asn Gly 280 285

Lys Pro Ser Gly Ala Arg Val Pro Ile Ser Glu 295 300

Leu Ala Met His Trp Thr Leu Ala Thr Met Phe 310 315 320

Lys Asp For Val Asn Met Leu Val Tyr Phe Leu Val Ser 325 330 335

Cys Gly Asn Leu Ala Ile Val Phe Ser Leu As His 340 345 350

Pro Val Ile Ser Lys Glu Glu Ala Val Asp Met Asp Phe 360 365

Gin Ile Thr Gly Arg Asp Val His Pro Gly Leu Phe 375 380

Phe Thr Gly Gly Leu Asn Tyr Gin His His Leu 390 395 400

Met Pro Arg His Asn Phe Ser Lys Ile Gin For Ala Val 405 410 415

Cys Lys Lys Tyr His Thr Thr Gly Met 420 425 430

Thr Ala Glu Val Phe Ser Arg Leu Asn Glu Val Ser Lys 440,445

Lys Met Gly Lys Ala Gin 455

109 (2) Information about SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 1488 pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: one year (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: DNA (genomic) (xi) Description SEQ ID NO: 3:

GTCCCCTGTC GCTGTCGGCA CACCCCATCC TCCCTCGCTC CCTCTGCGTT TGTCCTTGGC 60

CCACCGTCTC TCCTCCACCC TCCGAGACGA CTGCAACTGT AATCAGGAAC CGACAAATAC 120

ACGATTTCTT TTTACTCAGC ACCAACTCAA AATCCTCAAC CGCAACCCTT TTTCAGGATG 190

GCACCTCCCA ACACTATCGA TGCCGGTTTG ACCCAGCGTC ATATCAGCAC CTCGGCCCCA 240

AACTCGGCCA AGCCTGCCTT CGAGCGCAAC TACCAGCTCC CCGAGTTCAC CATCAAGGAG 300

ATCCGAGAGT GCATCCCTGC CCACTGCTTT GAGCGCTCCG GTCTCCGTGG TCTCTGCCAC 360

GTTGCCATCG ATCTGACTTG GGCGTCGCTC TTGTTCCTGG CTGCGACCCA GATCGACAAG 420

TTTGAGAATC CCTTGATCCG CTATTTGGCC TGGCCTGTTT ACTGGATCAT GCAGGGTATT 480

GTCTGCACCG GTGTCTGGGT GCTGGCTCAC GAGTGTGGTC ATCAGTCCTT CTCGACCTCC 540

AAGACCCTCA ACAACACAGT TGGTTGGATC TTGCACTCGA TGCTCTTGGT CCCCTACCAC 600

TCCTGGAGAA TCTCGCACTC GAAGCACCAC AAGGCCACTG GCCATATGAC CAAGGACCAG 660

GTCTTTGTGC CCAAGACCCG CTCCCAGGTT GGCTTGCCTC CCAAGGAGAA CGCTGCTGCT 720

GCCGTTCAGG AGGAGGACAT GTCCGTGCAC CTGGATGAGG AGGCTCCCAT TGTGACTTTG 780

TTCTGGATGG TGATCCAGTT CTTGTTCGGA TGGCCCGCGT ACCTGATTAT GAACGCCTCT 840

110

GGCCAAGACT CGCAACTTTT ACGGCCGCTG GACCTCGCAC TTCCACACGT ACTCGCCCAT TCGACATTAT TATCTCGGAC CTCGGTGTGT TGGCTGCCCT ATCTATGCCT CCATGCAGTT GTCGCTCTTG ACCGTCACCA AGTACTATAT CTCTTTGTCA ACTTTTGGTT GGTCCTGATC ACCTTCTTGC AGCACACCGA CCCCATTACC GCGAGGGTGC CTGGAATTTC CAGCGTGGAG CTCTTTGCAC TCGTTTGGCA AGTTCTTGGA CCATATGTTC CACGGCATTG TCCACACCCA CACTTGTTCT CGCAAATGCC GTTCTACCAT GCTGAGGAAG CTACCTATCA CTGCTGGGAG AGTACTATGT GTACGACCCA TCCCCGATCG TCGTTGCGGT TTCCGTGAGT GCCGATTCGT GGAGGATCAG GGAGACGTGG TCTTTTTCAA AAAAGACAAT GGACCACACA CAACCTTGTC TCTACAGACC TACGTATCAT CACTTCATAA AAGAACATGA GCTCTAGAGG CGTGTCATTC GCGCCTCC (2) SEQ ID NO: 4 information: (and .) SEQUENCE CHARACTERISTICS:

• · · · • · · * · · » « • · CTTTGAGCCC 900 CGGTGCCCTG 960 TGTCCCCTAC 1020 TCCCAAGCTG 1080 CGTTGACCGC 1140 TGTGGCCCAT 1200 TCTCAAGAAA 1260 CTGGAGGTCG 1320 GAAGTAAAAA 1380 GTAGCCATAC 1440 1488

(A) LENGTH: 399 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: peptide

(xi) Description sequence: SEQ ID NO: ' 4: Met Ala 1 Pro Pro Asn Thr Ile 5 Asp Ala Gly Leu Thr Gin Arg His Ile Ser Thr Ser Ala Pro Asn Ser Ala Lys For Ala 25 Phe Glu Arg Asn Tyr 30. Gin Leu For Glu 35 Phe Thr Ile Lys Glu Ile Arg Glu Cys Ile Pro Ala His Cys Phe Glu Arg Ser Gly Leu Arg Gly Leu Cys His Val Ala Xle Asp Leu Thr Trp Ala Ser Leu Leu Phe Leu Al 75 Ala Thr Gin Ile Asp Lys Phe Glu Asn For Leu Ile 85 Arg Tyr Leu Ala Trp Pro Val Tyr Ile Met Gin Gly Ile Val Cys Thr Gly Val Trp Val Leu Ala His Glu Cys Gly His Gin Ser Phe Ser Thr Ser Lys Thr 120 Leu Asn Asn Thr Val 125 Gly Trp Ile Leu His Ser Met Leu Leu Val Pro Tyr His Ser Trp Arg Ile Ser His Ser Lys His His Lys Ala Thr Gly 156 His Met Thr Lys Asp 160

• · · · 0 0 ·· ··· · ··· 0 · 00 0 ··· · 00 * ··

Gin Wall Phe Wall For 165 Lys Thr Arg Ser Gin Val Gly Leu For For 175 Lys Glu Asn Ala Ala Ala Ala Wall Gin Glu Glu Asp Met Ser Wall His Leu 180 185 190 Asp Glu Glu Ala For Ile Wall Thr Leu Phe Trp Met Wall Ile Gin Phe 195 200 205 Leu Phe Gly Trp For Ala Tyr Leu Ile Met Asn Ala Ser Gly Gin Asp 210 215 220 Tyr Gly Arg Trp Thr Ser His Phe His Thr Tyr Ser For Ile Phe Glu 225 230 235 240 For Arg Asn Phe Phe Asp Ile Ile Ile Ser Asp Leu Gly Wall Leu Ala 245 250 255 Ala Leu Gly Ala Leu Ile Tyr Ala Ser Met Gin Leu Ser Leu Leu Thr 260 265 270 Wall Thr Lys Tyr Tyr Ile Wall For Tyr Leu Phe Wall Asn Phe Trp Leu 275 280 285 Wall Leu Ile Thr Phe Leu Gin His Thr Asp For Lys Leu For His Tyr 290 295 300 Arg Glu Gly Ala Trp Asn Phe Gin Arg Gly Ala Leu Cys Thr Wall Asp 305 310 315 320 Arg Ser Phe Gly Lys Phe Leu Asp His Met Phe His Gly Ile Wall His 325 330 335 Thr His Wall Ala His His Leu Phe Ser Gin Met For Phe Tyr His Ala 340 345 350 Glu Glu Ala Thr Tyr His Leu Lys Lys Leu Leu Gly Glu Tyr Tyr Wall 355 360 365 Tyr Asp For Ser For Ile Wall Wall Ala Wall Trp Arg Ser Phe Arg Glu 370 375 380 Cys Arg Phe Wall Glu Asp Gin Gly Asp Wall Wall Phe Phe Lys Lys 385 390 395

2) Information about SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 355 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: peptide (xi) Description of sequence: SEQ ID NO: 5:

112

· · · * * * • •

Val Arg Lys Leu Arg Thr Leu Phe Gin Ser Leu Gly Tyr Tyr Asp 5 10 15 Dec ! Ser Ser Lys Ala Tyr Ser Lys Val Ser Phe 20 25 Asn Leu Cys Ile Trp Gly Leu Ser Thr Val Ala Lys Trp Gly 40 Gin Thr Ser Thr Leu Ala Asn 50 Val Leu Ser Ala Ala Leu Gly Leu 55 60 Phe Trp Cys Gly Trp Leu Ala His Asp Phe Leu Val Phe Gin Asp 80 Arg Phe Trp Gly Asp Leu Phe Gly Al Phe Leu Gly 90 Gly Val Cys Gin Gly Phe Ser Ser Ser 100 ALIGN! Lys Asp Lys His Asn 105 Thr His His Ala Ala Pro Asn Val His Gly Glu Asp Asp Asp 120 Asp Thr His Pro Leu Leu Thr Trp Ser Glu His Ala Leu Glu Met Phe Ser 136 140 Asp Val Pro Asp Glu Glu Leu Thr Arg Met Trp Ser Arg Phe Met Val Leu Asn Gin Thr 161 Php Tyr Phe Pro Ile Leu Ser Phe Ala Arg Leu Ser Trp Cys Leu Gin Ser Ile Leu Phe 180 Val Leu Pro Asn Gly Gin Ala His Lys Pro Ser 190 Gly Ala Arg 195 Val Pro Ile Ser Leu Val Glu Gin Leu Ser Leu Ala Met 205 His Trp Thr 210 Trp Tyr Leu Ala Thr Met Phe Ile Lys Asp Pro Val Asn Met Leu Val Tyr Phe Leu Val Ser Gin Ala 235 235 Val Cys Gly Asn 240 Leu Leu Ala Ile Val 245 Phe Ser Leu Asn His Asn Gly 250 Met Pro Val Ile 255 Ser Lys Glu Glu Ala 260 Val Asp Met Phe Phe Thr 265 Lys Gin Ile Ile Thr Gly Arg Asp Val His Pro Gly Leu Phe Ala Asn 280 Trp Phe Thr Gly 285 Gly Leu Asn Tyr Gin Ile Glu His His Leu Phe Pro 295 300 Ser Met Pro Arg His Asn Phe 305 Ser Lys Ile Gin Pro Ala Val Glu Thr 310 315 Leu Cys Lys Lys Asr Val Arg Tyr 325 His Thr Thr Gly Met Gly Thr Ala Glu 335 Val Phe Ser Arg Leu Asn Glu Val Ser Ser Lys Met Gly

340 345 350

Lys Ala Gin

113 ·· · · · 999 9 · ·· · · · · 9 * • · · · · ····· * «• · · · ·» · ·· ····· _and ♦ »,, (2 ) Information ο SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 104 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: linear MOLECULAR TYPE: peptide

(xi ) p opi s s eq enc E: SEQ ID NO : 6 Wall Thr Leu Tyr Thr Leu Ala Phe Wall Ala Ala Asn Ser Leu Gly Wall 1 5 10 15 Dec Leu Tyr Gly Wall Leu Ala Cys For Ser Wall Xaa For His Gin Ile Ala 20 May 25 30 Ala Gly Leu Leu Gly Leu Leu Trp Ile Gin Ser Ala Tyr Ile Gly Xaa 35 40 45 Asp Ser Gly His Tyr Wall Ile Met Ser Asn Lys Ser Asn Asn Xaa Phe 50 55 60 Ala Gin Leu Leu Ser Gly Asn Cys Leu Thr Gly Ile Ile Ala Trp Trp 65 70 75 80 Lys Trp Thr His Asn Ala His His Leu Ala Cys Asn Ser Leu Asp Tyr 85 90 95 Gly For Asn Leu Gin His Ile For

100 (2) Information about SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH .: 252 amino acids (B) TYPE: amino acid (C) SPECIES CHAIN: not relevant (D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: peptide (xi) Sequence description: SEQ ID NO: 7:

114

Gly Val Leu Tyr

Ile Ala Ala Ala Leu Leu 20

Gly His Asp Ser

Arg Phe Ala Gin Leu Leu Ser Gly 50

Ala Trp Trp Lys Trp Thr His Asn

Leu Asp Tyr Asp

Thr Lys Phe Phe Ser

Thr Phe Gly Pro Val Ala Arg

Tyr Tyr Pro Val Asn Cys Phe Gly 130

Phe Leu Leu Phu Phu Ser Lys Arg 145 150

Phe Ala Gly Phle Trp 165

Cys Leu For As Trp Pro Glu Arg 180

Thr Val Thr Ala Leu Gin His Ile 195 200

Ala Asp Val Tyr Val Gly Pro Pro 215 215

Gin Ala Ala Gly Thr

Phe Phe Gly Leu Gin Phe Gin 245

Cys Thr Ser Val Phe Ala His Gin 10

Leu Trp Ile Gin Ser

Ile Met Ser Asn Lys Ser Tyr Asn

Asn Cys Leu Thr Gly Ile

Ala His His Leu Ala Cys Asn Ser 75

His Ile Pro Val Phe

Ser Arg Phe Tyr

Leu Val Ser Tyr Gin His Phe Thr 126

Arg Ile Asn Leu Phe Ile Gin Thr 139

Glu Val Pro Asp Arg

Thr Trp Phe Leu Leu Val Ser 170 175

Phe Phe Phr Thr Ser Phe 185 190

Gin Phe Thr Leu Asn His Phe Al 205

Thr Gly Ser Trp Phe Glu Lys 220

Ser Cys Arg. Ser Tyr Met Asp Trp 235 240

Leu Glu His His 250

115 (2) Information about SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 125 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: peptide (xi) Sequence description: SEQ ID NO: 8:

Gly 1 Xaa Xaa Asn Phe 5 Ala Gly Ile Leu Wall 10 Phe Trp Thr Trp Phe 15 Dec For Leu Leu Wall Ser Cys Leu For Asn Trp For Glu Arg Phe Xaa Phe Wall 20 May 25 30 Phe Thr Gly Phe Thr Wall Thr Ala Leu Gin His Ile Gin Phe Thr Leu 35 40 45 Asn His Phe Ala Ala Asp Wall Tyr Wall Gly For For Thr Gly Ser Asp 50 55 60 Trp Phe Glu Lys Gin Ala Ala Gly Thr Ile Asp Ile Ser Cys Arg Ser 65 70 75 80 Tyr Met Asp Trp Phe Phe Cys Gly Leu Gin Phe Gin Leu Glu His His 85 90 95 Leu Phe For Arg Leu For Arg Cys His Leu Arg Lys Wall Ser For Wall 100 ALIGN! 105 110 Gly Gin Arg Gly Phe Gin Arg Lys Xaa Asn Leu Ser Xaa 115 120 125

9

999 ··

9

116 • · * (2) Information about SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 131 amino acids

(B ) TYPE: amino acid (C ) CHAIN TYPE: not relevant (D ) TOPOLOGY: linear (ii) SPECIES MOLECULES: peptide (Xi) Description SEQ ID NO: 9:

Pro Ala Thr Glu Glu Glu Glu Leu Ala Trp Met Ile Thr Phe Tyr Val

5 10 15

Arg Phe Phu Ler Thr Val Pro Leu Leu Gly Leu Lys Ala Phe Leu

25 30

Gly Leu Phe Phe Ile Val Arg Phe Leu Glu Ser Phn Val Trp 35 40 45

Val Thr Gin As His His Ile For His His Asle Arg Asn 50 55 60

Met Asp Trp Val Ser Thr Gin Leu Gin Ala Thr Cys Asn Val His.Lys 65 70 75 80

Ser Ala Phe Asn As Trp Phe Ser Gly His Leu Asn Phe Gin Ile Glu 85 90 95

His His Leu Phe Thr Met Pro Arg His Asn Tyr His Xaa Val Ala 100 105 110

Pro Leu Val Gin Ser Leu Cys Ala Lys His Gly Ile Glu Tyr Gin Ser 115 120 125

Lys Pro Leu 130 (2) Information about SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 87 amino acids (B) TYPE: amino acid • · · ·

117 (C) CHAIN TYPE: not relevant (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: peptide

(xi) Description sequence: SEQ ID NO: ' 10: Cys 1 Ser For Lys Ser 5 Ser For Thr Arg Asn 10 Met Thr For Ser For 15 Dec Phe Ile Asp Trp Leu 20 May Trp Gly Gly Leu Asn 25 Tyr Gin Ile Glu His 30 His Leu Phe For Thr 35 Met For Arg Cys Asn 40 Leu Asn Arg Cys Met 45 Lys Tyr Wall Lys Glu 50 Trp Cys Ala Glu Asn 55 Asn Leu For Tyr Leu 60 Wall Asp Asp Tyr Phe 65 Wall Gly Tyr Asn Leu 70 Asn Leu Gin Gin Leu 75 Lys Asn Met Ala Glu 80 Leu Wall Gin Ala Lys 85 Ala Ala Information about SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 143 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: peptide

(xi ) p opi S S eq enc E: SEQ ID NO : i: 1: Arg His Glu Ala Ala Arg Gly Gly Thr Arg Leu Ala Tyr Met Leu Wall 1 5 10 15 Dec Cys Met Gin Trp Thr Asp Leu Leu Trp Ala Ala Ser Phe Tyr Ser Arg 20 May 25 30 Phe Phe Leu Ser Tyr Ser For Phe Tyr Gly Ala Thr Gly Thr Leu Leu 35 40 45 Leu Phe Wall Ala Wall Arg Wall Leu Glu Ser His Trp Phe Wall Trp Ile 50 55 60 Thr Gin Met Asn His Ile For Lys Glu Ile Gly His Glu Lys His Arg 65 70 75 80 Asp Trp Ala Ser Ser Gin Leu Ala Ala Thr Cys Asn Wall Glu For Ser 85 90 95 Leu Phe Ile Asp Trp Phe Ser Gly His Leu Asn Phe Gin Ile Glu His 100 ALIGN! 105 110

118

His Leu Phe For Thr Met Thr Arg His Asn Tyr Arg 120 Xaa Val Ala Pro Leu Wall Lys Ala Phe Cys Ala Lys His Gly Leu His Tyr Glu Wall 130 135 140

(2) SEQ ID NO: 12 information:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (xi) Sequence description: SEQ ID NO: 12:

CCAAGCTTCT GCAGGAGCTC τττΤΤΤΤΤΤΤ TTTTT 35 (2) Information about SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 nucleic acids (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (xi) Sequence description: SEQ ID NO: 13:

CUACUACUAC UAGGAGTCCT CTACGGTGTT TTG 33 (2) Information about SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 33 pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear

«· ·

119 (ii) MOLECULAR TYPE: other nucleic acid (xi) Sequence description: SEQ ID NO: 14 ':

CAUCAUCAUC AUATGATGCT CAAGCTGAAA CTG 33 (2) Information about SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 bp (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (xi) Sequence description: SEQ ID NO: 15:

TACCAACTCG AGAAAATTGGC TGCTGCTCCC AGTGTGAGG 39 (2) Information about SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 bp (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (xi) Sequence description: SEQ ID NO: 16:

AACTGATCTA GATTACTGCG CCTTACCCAT CTTGGAGGC 39 (2) Information about SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 pairs (b) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid

120 (xi) Description of sequence: SEQ ID NO: 17:

TACCAACTCG AGAAAATGGC ACCTCCCAAC ACTATCGAT 39 (2) Information about SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 39 pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (xi) Description SEQ ID NO: 18:

AACTGATCTA GATTACTTCT TGAAAAAGAC CACGTCTCC 39 (2) Information about SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 746 nucleic acids (B) TYPE: nucleic acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: nucleic acid (xi) Sequence description: SEQ ID NO: 19:

CGTATGTCAC TCCATTCCAA ACTCGTTCAT GGTATCATAA ATATCAACAC ATTTACGCTC 60 CACTCCTCTA TGGTATTTAC ACACTCAAAT ATCGTACTCA AGATTGGGAA GCTTTTGTAA 120 AGGATGGTAA AAATGGTGCA ATTCGTGTTA GTGTCGCCAC AAATTTCGAT AAGGCCGCTT 180 ACGTCATTGG T.AAATTGTCT TTTGTTTTCT TCCGTTTCAT CCTTCCACTC CGTTATCATA 240 GCTTTACAGA TTTAATTTGT TATTTCCTCA TTGCTGAATT CGTCTTTGGT TGGTATCTCA 300 CAATTAATTT CCAAGTTAGT CATGTCGCTG AAGATCTCAA ATTCTTTGCT ACCCCTGAAA 360 GACCAGATGA ACCATCTCAA ATCAATGAAG ATTGGGCAAT CCTTCAACTT AAAACTACTC 420 AAGATTATGG TCATGGTTCA CTCCTTTGTA CCTTTTTTAG TGGTTCTTTA AATCATCAAG 480 TTGTTCATCA TTTATTCCCA TCAATTGCTC AAGATTTCTA CCCACAACTT GTACCAATTG 540 TAAAAGAAGT TTGTAAAGAA CATAACATTA CTTACCACAT TAAACCAAAC TTCACTGAAG 600 CTATTATGTC ACACATTAAT TACCTTTACA AAATGGGTAA TGATCCAGAT TATGTTAAAA 660 AACCATTAGC CTCAAAAGAT GATTAAATGA AATAACTTAA AAACCAATTA TTTACTTTTG 720

ACAAACAGTA ATATTAATAA ATACAA

746

(2) Information about SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 227 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: peptide (xi) Sequence description: SEQ ID NO: 20:

Tyr Wall Thr For Phe Gin Thr Arg Ser Trp Tyr His Lys Tyr Gin 1 5 10 15 Dec His Ile Tyr Ala For Leu Leu Tyr Gly Ile Tyr Thr Leu Lys Tyr 20 May 25 30 Arg Thr Gin Asp Trp Glu Ala Phe Wall Lys Asp Gly Lys Asn Gly 35 40 45 Ala Ile Arg Wall Ser Wall Ala Thr Asn Phe Asp Lys Ala Ala Tyr 50 55 60 Wall Ile Gly Lys Leu Ser Phe Wall Phe Phe Arg Phe Ile Leu For 65 70 75 Leu Arg Tyr His Ser Phe Thr Asp Leu Ile Cys Tyr Phe Leu Ile 80 85 90 Ala Glu Phe Wall Phe Gly Trp Tyr Leu Thr Ile Asn Phe Gin Wall 95 100 ALIGN! 105 Ser His Wall Ala Glu Asp Leu Lys Phe Phe Ala Thr For Glu Arg 110 115 120 For Asp Glu For Ser Gin Ile Asn Glu Asp Trp Ala Ile Leu Gin 125 130 135 Leu Lys Thr Thr Gin Asp Tyr Gly His Gly Ser Leu Leu Cys Thr 140 145 150 Phe Phe Ser Gly Ser Leu Asn His Gin Wall Wall His His Leu Phe 155 160 165 For Ser Ile Ala Gin Asp Phe Tyr For Gin Leu Wall For Ile Wall 170 175 180 Lys Glu Wall Cys Lys Glu His Asn Ile Thr Tyr His Ile Lys For 185 190 195 Asn Phe Thr Glu Ala Ile Met Ser His Ile Asn Tyr Leu Tyr Lys 200 205 210 Met Gly Asn Asp For Asp Tyr Wall Lys Lys For Leu Ala Ser Lys 215 220 225 Asp Asp * * *

(2) SEQ ID NO: 21 information:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 494 nucleic acids (B) TYPE: nucleic acid (C) CHAIN TYPE: not relevant

122

(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: nucleic acid (xi) Description of sequence: SEQ ID NO: 21:

TTTTGGAAGG NTCCAAGTTN ACCACGGANT NGGCAAGTTN ACGGGGCGGA AANCGGTTTT 60

CCCCCCAAGC CTTTTGTCGA CTGGTTCTGT GGTGGCTTCC AGTACCAAGT CGACCACCAC 120

TTATTCCCCA GCCTGCCCCG ACACAATCTG GCCAAGACAC ACGCACTGGT CGAATCGTTC 180

TGCAAGGAGT GGGGTGTCCA GTACCACGAA GCCGACCTCG TGGACGGGAC CATGGAAGTC 240

TTGCACCATT TGGGCAGCGT GGCCGGCGAA TTCGTCGTGG ATTTTGTACG CGACGGACCC 300

GCCATGTAAT CGTCGTTCGT GACGATGCAA GGGTTCACGC.ACATCTACAC ACACTCACTC 360 ACACAACTAG TGTAACTCGT ATAGAATTCG GTGTCGACCT GGACCTTGTT TGACTGGTTG 420 GGGATAGGGT AGGTAGGCGG ACGCGTGGGT CGNCCCCGGG AATTCTGTGA CCGGTACCTG 480 GCCCGCGTNA AAGT ⁴⁹⁴ (2) Information for SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 87 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: peptide

(Xi) ¹ Description sequence SEQ ID NO: 22: Phe Trp Lys Xxx For Ser Xxx For Arg Xxx Xxx Gin Val Xxx Gly 1 5 10 15 Dec Ala Glu Xxx Gly Phe Pro Pro Lys Pro Phe Wall Asp Trp Phe Cys 20 May 25 30 Gly Gly Phe Gin Tyr Gin Val Asp His His Leu Phe Pro Ser Leu 35 40 45 For Arg His Asn Leu Ala Lys Thr His Ala Leu Val Glu 50 55 60 Cys Lys Glu Trp Gly Val Gin Tyr His Glu Ala Asp. Leu Val Asp 65 70 75 Gly Thr Met Glu Val Leu His His Leu Gly Ser Val Glu Glu 65 70 75 Phe Wall Val Asp Phe Val Arg Asp Gly For Ala Met 80 85

(2) Information about SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 520 nucleic acids (B) TYPE: amino acid

(C) CHAIN TYPE: not relevant (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: nucleic acid (xi) Sequence description: SEQ ID NO: 23:

GGATGGAGTT CGTCTGGATC GCTGTGCGCT ACGCGACGTG GTTTAAGCGT CATGGGTGCG 60 CTTGGGTACA CGCCGGGGCA GTCGTTGGGC ATGTACTTGT GCGCCTTTGG TCTCGGCTGC 120 ATTTACATTT TTCTGCAGTT CGCCGTAAGT CACACCCATT TGCCCGTGAG CAACCCGGAG 180 GATCAGCTGC ATTGGCTCGA GTACGCGCGG ACCACACTGT GAACATCAGC ACCAAGTCGT 240 GGTTTGTCAC ATGGTGGATG TCGAACCTCA ACTTTCAGAT CGAGCACCAC CTTTTCCCCA 300 CGGCGCCCCA GTTCCGTTTC AAGGAGATCA GCCCGCGCGT CGAGGCCCTC TTCAAGCGCC 360 ACGGTCTCCC TTACTACGAC ATGCCCTACA CGAGCGCCGT CTCCACCACC TTTGCCAACC 420 TCTACTCCGT CGGCCATTCC GTCGGCGACG CCAAGCGCGA CTAGCCTCTT TTCCTAGACC 480 TTAATTCCCC ACCCCACCCC ATGTTCTGTC TTCCTCCCGC 520 (2) SEQ ID NO: 24 info:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 153 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: peptide

( (xi) Description sequence SEQ ID NO: 24: Met 1 Glu Phe Val Ip Ala 5 Val Arg Tyr 10 Ala Thr Trp Phe Lys Arg His Gly Cys Ala Trp Val His Ala Gly 25 Ala Val Val Gly His Wall Leu Val Arg Leu Trp Ser 34 Arg Leu His 40 Leu His Phe Ser Ala Wall Arg Arg Lys Ser His Pro Phe Ala Arg 55 Glu Gin Pro Gly Gly Ser Ala Ala Leu Ala Arg Val Arg Ala Asp 70 His Thr Val Asn Ile Ser Thr Lys Ser Trp Phe Val 80 Thr Trp Trp 85 Met Ser Asn Leu Asn 90 Phe Gin Ile Glu His His Leu Phe Pro Thr 100 ALIGN! Ala For Gin Phe Arg 105 Phe Lys Glu Ile Ser Pro Arg Val Glu Ala 115 Leu Phe Lys Arg His 120 Gly Leu Pro Tyr Tyr Asp Met Pro Tyr Thr 130 Ser Ala Val Ser Thr Thr Phe Ala Asn Leu Tyr Ser 140 Val Gly His 145 Ser Val Gly Asp Ala

Lys Arg Asp »

124

2) Information about SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 420 nucleic acids (B) TYPE: nucleic acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: nucleic acid (xi) Sequence description: SEQ ID NO: 25:

ACGCGTCCGC CCACGCGTCC GCCGCGAGCA ACTCATCAAG GAAGGCTACT TTGACCCCTC 60 GCTCCCGCAC ATGACGTACC GCGTGGTCGA GATTGTTGTT CTCTTCGTGC TTTCCTTTTG 120 GCTGATGGGT CAGTCTTCAC CCCTCGCGCT CGCTCTCGGC ATTGTCGTCA GCGGCATCTC 180 TCAGGGTCGC TGCGGCTGGG TAATGCATGA GATGGGCCAT GGGTCGTTCA CTGGTGTCAT 240 TTGGCTTGAC GACCGGTTGT GCGAGTTCTT TTACGGCGTT GGTTGTGGCA TGAGCGGTCA 300 TTACTGGAAA AACCAGCACA GCAAACACCA CGCAGCGCCA AACCGGCTCG AGCACGATGT 360 AGATCTCAAC ACCTTGCCAT TGGTGGCCTT CAACGAGCGC GTCGTGCGCA AGGTCCGACC 420

2) Information about SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 125 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: peptide (xi) Sequence description: SEQ I

NO: 26:

Arg 1 Wall Arg For Arg 5 Tyr Phe Asp For Ser 20 May Ile Wall Wall Leu Phe 35 Ser For Leu Ala Leu 50 Gin Gly Arg Cys Gly 65 Phe Thr Gly Wall Ile 65 Tyr Gly Wall Gly Cys 80 His Ser Lys His His 95 Asp Leu Asn Thr Leu 110 Arg Lys Wall Arg For 125

Wall Arg Arg Glu Gin 10 Leu For His Met Thr 25 Wall Leu Ser Phe Trp 40 Ala Leu Gly Ile Wall 55 Trp Wall Met His Glu 70 Trp Leu Asp Asp Arg 70 Gly Met Ser Gly His 85 Ala Ala For Asn Arg 100 ALIGN! For Leu Wall Ala Phe 115

Leu Ile Lys Glu Gly 15 Dec Tyr Arg Wall Wall Glu 30 Leu Met Gly Gin Ser 45 Wall Ser Gly Xle Ser 60 Met Gly His Gly Ser 75 Leu Cys Glu Phe Phe 75 Tyr Trp Lys Asn Gin 90 Leu Glu His Asp Wall 105 Asn Glu Arg Wall Wall 120

«·

125 (2) Information about SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1219 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (edited contig 2692004)

(xi) Description sequence: AGATCCTGGC SEQ ID AAAGTATCCA NO: 27: GCACGCCGAC CGGCGCCGGG GAGATAAAGT CCTTGATGAA 60 ACCTGATCCC AATTTGATAT GGATTATAAT TATGATGGTT CTCACCCAGT TGGGTGCATT 120 TTACATAGTA AAAGACTTGG ACTGGAAATG GGTCATATTT GGGGCCTATG CGTTTGGCAG 180 TTGCATTAAC CACTCAATGA CTCTGGCTAT TCATGAGATT GCCCACAATG CTGCCTTTGG 240 CAACTGCAAA GCAATGTGGA ATCGCTGGTT TGGAATGTTT GCTAATCTTC CTATTGGGAT 300 TCCATATTCA ATTTCCTTTA AGAGGTATCA CATGGATCAT CATCGGTACC TTGGAGCTGA 360 TGGCGTCGAT GTAGATATTC CTACCGATTT TGAGGGCTGG TTCTTCTGTA CCGCTTTCAG 420 AAAGTTTATA TGGGTTATTC TTCAGCCTCT CTTTTATGCC TTTCGACCTC TGTTCATCAA 480 CCCCAAACCA ATTACGTATC TGGAAGTTAT CAATACCGTG GCACAGGTCA CTTTTGACAT 540 TTTAATTTAT TACTTTTTGG GAATTAAATC CTTAGTCTAC ATGTTGGCAG CATCTTTACT 600 TGGCCTGGGT TTGCACCCAA TTTCTGGACA TTTTATAGCT GAGCATTACA TGTTCTTAAA 660 GGGTCATGAA ACTTACTCAT ATTATGGGCC TCTGAATTTA CTTACCTTCA ATGTGGGTTA 720 TCATAATGAA CATCATGATT TCCCCAACAT TCCTGGAAAA AGTCTTCCAC TGGTGAGGAA 780 AATAGCAGCT GAATACTATG ACAACCTCCC TCACTACAAT TCCTGGATAA AAGTACTGTA 840 TGATTTTGTG ATGGATGATA CAATAAGTCC CTACTCAAGA ATGAAGAGGC ACCAAAAAGG 900 AGAGATGGTG CTGGAGTAAA TATCATTAGT GCCAAAGGGA TTCTTCTCCA AAACTTTAGA 960 TGATAAAATG GAATTTTTGC ATTATTAAAC TTGAGACCAG TGATGCTCAG AAGCTCCCCT 1020 GGCACAATTT CAGAGTAAGA GCTCGGTGAT ACCAAGAAGT GAATCTGGCT TTTAAACAGT 1080 CAGCCTGACT CTGTACTGCT CAGTTTCACT CACAGGAAAC TTGTGACTTG TGTATTATCG 1140 TCATTGAGGA TGTTTCACTC ATGTCTGTCA TTTTATAAGC ATATCATTTA AAAAGCTTCT 1200

AAAAAGCTAT TTCGCCAGG

1219

126 (2) Information about SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 65 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (edited contig 2153526) (xi) Sequence description: SEQ ID NO: 28:

TTACCTTCTA CGTCCGCTTC TTCCTCACTT ATGTGCCACT ATTGGGGCTG AAAGCTTCCT 60 GGGCCTTTTC TTCATAGTCA GGTTCCTGGA AAGCAACTGG TTTGTGTGGG TGACACAGAT 120 GAACCATATT CCCATGCACA TTGATCATGA CCGGAACATG GACTGGGTTT CCACCCAGCT 180 CCAGGCCACA TGCAATGTCC ACAAGTCTGC CTTCAATGAC TGGTTCAGTG GACACCTCAA 240 CTTCCAGATT GAGCACCATC TTTTTCCCAC GATGCCTCGA CACAATTACC ACAAAGTGGC 300 TCCCCTGGTG CAGTCCTTGT GTGCCAAGCA TGGCATAGAG TACCAGTCCA AGCCCCTGCT 360 GTCAGCCTTC GCCGACATCA TCCACTCACT AAAGGAGTCA GGGCAGCTCT GGCTAGATGC 420 CTATCTTCAC CAATAACAAC AGCCACCCTG CCCAGTCTGG AAGAAGAGGA GGAAGACTCT 480 GGAGCCAAGG CAGAGGGGAG CTTGAGGGAC AATGCCACTA TAGTTTAATA CTCAGAGGGG 540 GTTGGGTTTG GGGACATAAA GCCTCTGACT CAAACTCCTC CCTTTTATCT TCTAGCCACA 600

GTTCTAAGAC CCAAAGTGGGGGGTGGACAC AGAAGTCCCT AGGAGGGAAG GAGCT

655

127 * · (2) Information about SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 304 bp (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other contig 3506132 nucleic acid (edited (xi)

Sequence Description: SEQ ID NO: 29:

GTCTTTTACT TTGGCAATGG CTGGATTCCT ACCCTCATCA CGGCCTTTGT CCTTGCTACC 60 TCTCAGGCCC AAGCTGGATG GCTGCAACAT GATTATGGCC ACCTGTCTGT CTACAGAAAA 120 CCCAAGTGGA ACCACCTTGT CCACAAATTC GTCATTGGCC ACTTAAAGGG TGCCTCTGCC 180 AACTGGTGGA ATCATCGCCA CTTCCAGCAC CACGCCAAGC CTAACATCTT CCACAAGGAT 240 CCCGATGTGA ACATGCTGCA CGTGTTTGTT CTGGGCGAAT GGCAGCCCAT CGAGTACGGC 300

AAGA ³⁰⁴ (2) Information about SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 918 pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: other nucleic acid kontig 3854933) (xi) Description SEQ ID NO: 30:

'edited

128

CAGGGACCTA CCCCGCGCTA CTTCACCTGG GACGAGGTGG CCCAGCGCTC AGGGTGCGAG 60 GAGCGGTGGC TAGTGATCGA CCGTAAGGTG TACAACATCA GCGAGTTCAC CCGCCGGCAT 120 CCAGGGGGCT CCCGGGTCAT CAGCCACTAC GCCGGGCAGG ATGCCACGGA TCCCTTTGTG 180 GCCTTCCACA TCAACAAGGG CCTTGTGAAG AAGTATATGA ACTCTCTCCT GATTGGAGAA 240 CTGTCTCCAG AGCAGCCCAG CTTTGAGCCC ACCAAGAATA AAGAGCTGAC AGATGAGTTC 300 CGGGAGCTGC GGGCCACAGT GGAGCGGATG GGGCTCATGA AGGCCAACCA TGTCTTCTTC 360 CTGCTGTACC TGCTGCACAT CTTGCTGCTG GATGGTGCAG CCTGGCTCAC CCTTTGGGTC 420 TTTGGGACGT CCTTTTTGCC CTTCCTCCTC TGTGCGGTGC TGCTCAGTGC AGTTCAGGCC 480 CAGGCTGGCT GGCTGCAGCA TGACTTTGGG CACCTGTCGG TCTTCAGCAC CTCAAAGTGG 540 AACCATCTGC TACATCATTT TGTGATTGGC CACCTGAAGG GGGCCCCCGC CAGTTGGTGG 600 AACCACATGC ACTTCCAGCA CCATGCCAAG CCCAACTGCT TCCGCAAAGA CCCAGACATC 660 AACATGCATC CCTTCTTCTT TGCCTTGGGG AAGATCCTCT CTGTGGAGCT TGGGAAACAG 720 AAGAAAAAAT ATATGCCGTA CAACCACCAG CACARATACT TCTTCCTAAT TGGGCCCCCA 780 GCCTTGCTGC CTCTCTACTT CCAGTGGTAT ATTTTCTATT TTGTTATCCA GCGAAAGAAG 840 TGGGTGGACT TGGCCTGGAT CAGCAAACAG GAATACGATG AAGCCGGGCT TCCATTGTCC 900 ACCGCAAATG CTTCTAAA 918

(2) Information about SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1686 pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (edited contig 2511785) (xi) Sequence description: SEQ ID NO: 31:

• »

129 • · • · • · • · · • • · • • · GCCACTTAAA GGGTGCCTCT GCCAACTGGT GGAATCATCG CCACTTCCAG CACCACGCCA 60 AGCCTAACAT CTTCCACAAG GATCCCGATG TGAACATGCT GCACGTGTTT GTTCTGGGCG 120 AATGGCAGCC CATCGAGTAC GGCAAGAAGA AGCTGAAATA CCTGCCCTAC AATCACCAGC 180 ACGAATACTT CTTCCTGATT GGGCCGCCGC TGCTCATCCC CATGTATTTC CAGTACCAGA 240 TCATCATGAC CATGATCGTC CATAAGAACT GGGTGGACCT GGCCTGGGCC GTCAGCTACT 300 ACATCCGGTT CTTCATCACC TACATCCCTT TCTACGGCAT CCTGGGAGCC CTCCTTTTCC 360 TCAACTTCAT CAGGTTCCTG GAGAGCCACT GGTTTGTGTG GGTCACACAG ATGAATCACA 420 TCGTCATGGA GATTGACCAG GAGGCCTACC GTGACTGGTT CAGTAGCCAG CTGACAGCCA 480 CCTGCAACGT GGAGCAGTCC TTCTTCAACG ACTGGTTCAG TGGACACCTT AACTTCCAGA 540 TTGAGCACCA CCTCTTCCCC ACCATGCCCC GGCACAACTT ACACAAGATC GCCCCGCTGG 600 TGAAGTCTCT ATGTGCCAAG CATGGCATTG AATACCAGGA GAAGCCGCTA CTGAGGGCCC 660 TGCTGGACAT CATCAGGTCC CTGAAGAAGT CTGGGAAGCT GTGGCTGGAC GCCTACCTTC 720 ACAAATGAAG CCACAGCCCC CGGGACACCG TGGGGAAGGG GTGCAGGTGG GGTGATGGCC 780 AGAGGAATGA TGGGCTTTTG TTCTGAGGGG TGTCCGAGAG GCTGGTGTAT GCACTGCTCA 840 CGGACCCCAT GTTGGATCTT TCTCCCTTTC TCCTCTCCTT TTTCTCTTCA CATCTCCCCC 900 ATAGCACCCT GCCCTCATGG GACCTGCCCT CCCTCAGCCG TCAGCCATCA GCCATGGCCC 960 TCCCAGTGCC TCCTAGCCCC TTCTTCCAAG GAGCAGAGAG GTGGCCACCG GGGGTGGCTC 1020 TGTCCTACCT CCACTCTCTG CCCCTAAAGA TGGGAGGAGA CCAGCGGTCC ATGGGTCTGG 1080 CCTGTGAGTC TCCCCTTGCA GCCTGGTCAC TAGGCATCAC CCCCGCTTTG GTTCTTCAGA 1140 TGCTCTTGGG GTTCATAGGG GCAGGTCCTA GTCGGGCAGG GCCCCTGACC CTCCCGGCCT 1200 GGCTTCACTC TCCCTGACGG CTGCCATTGG TCCACCCTTT CATAGAGAGG CCTGCTTTGT 1260 TACAAAGCTC GGGTCTCCCT CCTGCAGCTC GGTTAAGTAC CCGAGGCCTC TCTTAAGATG 1320 TCCAGGGCCC CAGGCCCGCG GGCACAGCCA GCCCAAACCT TGGGCCCTGG AAGAGTCCTC 1380 CACCCCATCA CTAGAGTGCT CTGACCCTGG GCTTTCACGG GCCCCATTCC ACCGCCTCCC 1440 CAACTTGAGC CTGTGACCTT GGGACCAAAG GGGGAGTCCC TCGTCTCTTG TGACTCAGCA 1500 GAGGCAGTGG CCACGTTCAG GGAGGGGCCG GCTGGCCTGG AGGCTCAGCC CACCCTCCAG 1560 CTTTTCCTCA GGGTGTCCTG AGGTCCAAGA TTCTGGAGCA ATCTGACCCT TCTCCAAAGG 1620 CTCTGTTATC AGCTGGGCAG TGCCAGCCAA TCCCTGGCCA TTTGGCCCCA GGGGACGTGG 1680

GCCCTG

1686

130 (2) Information about SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1843 pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (kontig 2535) (xi) Sequence description: SEQ ID NO : 32:

GTCTTTTACT TTGGCAATGG CTGGATTCCT ACCCTCATCA CGGCCTTTGT CCTTGCTACC 60 TCTCAGGCCC AAGCTGGATG GCTGCAACAT GATTATGGCC ACCTGTCTGT CTACAGAAAA 120 CCCAAGTGGA ACCACCTTGT CCACAAATTC GTCATTGGCC ACTTAAAGGG TGCCTCTGCC 180 AACTGGTGGA ATCATCGCCA CTTCCAGCAC CACGCCAAGC CTAACATCTT CCACAAGGAT 240 CCCGATGTGA ACATGCTGCA CGTGTTTGTT CTGGGCGAAT GGCAGCCCAT CGAGTACGGC 300 AAGAAGAAGC TGAAATACCT GCCCTACAAT CACCAGCACG AATACTTCTT CCTGATTGGG 360 CCGCCGCTGC TCATCCCCAT GTATTTCCAG TACCAGATCA TCATGACCAT GATCGTCCAT 420 AAGAACTGGG TGGACCTGGC CTGGGCCGTC AGCTACTACA TCCGGTTCTT CATCACCTAC 480 ATCCCTTTCT ACGGCATCCT GGGAGCCCTC CTTTTCCTCA ACTTCATCAG GTTCCTGGAG 540 AGCCACTGGT TTGTGTGGGT CACACAGATG AATCACATCG TCATGGAGAT TGACCAGGAG 600 GCCTACCGTG ACTGGTTCAG TAGCCAGCTG ACAGCCACCT GCAACGTGGA GCAGTCCTTC 660 TTCAACGACT GGTTCAGTGG ACACCTTAAC TTCCAGATTG AGCACCACCT CTTCCCCACC 720 ATGCCCCGGC ACAACTTACA CAAGATCGCC CCGCTGGTGA AGTCTCTATG TGCCAAGCAT 780 GGCATTGAAT ACCAGGAGAA GCCGCTACTG AGGGCCCTGC TGGACATCAT CAGGTCCCTG 840 AAGAAGTCTG GGAAGCTGTG GCTGGACGCC TACCTTCACA AATGAAGCCA CAGCCCCCGG 900 GACACCGTGG GGAAGGGGTG CAGGTGGGGT GATGGCCAGA GGAATGATGG GCTTTTGTTC 960 TGAGGGGTGT CCGAGAGGCT GGTGTATGCA CTGCTCACGG ACCCCATGTT GGATCTTTCT 1020 CCCTTTCTCC TCTCCTTTTT CTCTTCACAT CTCCCCCATA GCACCCTGCC CTCATGGGAC 1080 CTGCCCTCCC TCAGCCGTCA GCCATCAGCC ATGGCCCTCC CAGTGCCTCC TAGCCCCTTC 1140 TTCCAAGGAG CAGAGAGGTG GCCACCGGGG GTGGCTCTGT CCTACCTCCA CTCTCTGCCC 1200 CTAAAGATGG GAGGAGACCA GCGGTCCATG GGTCTGGCCT GTGAGTCTCC CCTTGCAGCC 1260 TGGTCACTAG GCATCACCCC CGCTTTGGTT CTTCAGATGC TCTTGGGGTT CATAGGGGCA 1320 GGTCCTAGTC GGGCAGGGCC CCTGACCCTC CCGGCCTGGC TTCACTCTCC CTGACGGCTG 1380 CCATTGGTCC ACCCTTTCAT AGAGAGGCCT GCTTTGTTAC AAAGCTCGGG TCTCCCTCCT 1440 GCAGCTCGGT TAAGTACCCG AGGCCTCTCT TAAGATGTCC AGGGCCCCAG GCCCGCGGGC 1500

· · * * * * * • »» »

1560

1620

1680

1740

1800

1843

ACAGCCAGCC CAAACCTTGG GCCCTGGAAG AGTCCTCCAC CCCATCACTA GAGTGCTCTG ACCCTGGGCT TTCACGGGCC CCATTCCACC GCCTCCCCAA CTTGAGCCTG TGACCTTGGG accaaagggg gagtccctcg tctcttgtga ctcagcagag gcagtggcca CGTTCAGGGA GGGGCCGGCT GGCCTGGAGG CTCAGCCCAC CCTCCAGCTT TTCCTCAGGG TGTCCTGAGG

TCCAAGATTC TGGAGCAATC TGACCCTTCT CCAAAGGCTC TGTTATCAGC TGGGCAGTGC

CAGCCAATCC CTGGCCATTT GGCCCCAGGG GACGTGGGCC CTG (2) Information about SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2257 bp (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (edited contig 253538a)

(xi) Description sequence SEQ ID NO: 33: CAGGGACCTA CCCCGCGCTA CTTCACCTGG GACGAGGTGG CCCAGCGCTC AGGGTGCGAG 60 GAGCGGTGGC TAGTGATCGA CCGTAAGGTG TACAACATCA GCGAGTTCAC CCGCCGGCAT 120 CCAGGGGGCT CCCGGGTCAT CAGCCACTAC GCCGGGCAGG ATGCCACGGA TCCCTTTGTG 180 GCCTTCCACA TCAACAAGGG CCTTGTGAAG AAGTATATGA ACTCTCTCCT GATTGGAGAA 240 CTGTCTCCAG AGCAGCCCAG CTTTGAGCCC ACCAAGAATA AAGAGCTGAC AGATGAGTTC 300 CGGGAGCTGC GGGCCACAGT GGAGCGGATG GGGCTCATGA AGGCCAACCA TGTCTTCTTC 360 CTGCTGTACC TGCTGCACAT CTTGCTGCTG GATGGTGCAG CCTGGCTCAC CCTTTGGGTC 420 TTTGGGACGT CCTTTTTGCC CTTCCTCCTC TGTGCGGTGC TGCTCAGTGC AGTTCAGCAG 480 GCCCAAGCTG GATGGCTGCA ACATGATTAT GGCCACCTGT CTGTCTACAG AAAACCCAAG 540 TGGAACCACC TTGTCCACAA ATTCGTCATT GGCCACTTAA AGGGTGCCTC TGCCAACTGG 600 TGGAATCATC GCCACTTCCA GCACCACGCC AAGCCTAACA TCTTCCACAA GGATCCCGAT 660 GTGAACATGC TGCACGTGTT TGTTCTGGGC GAATGGCAGC CCATCGAGTA CGGCAAGAAG 720 AAGCTGAAAT ACCTGCCCTA CAATCACCAG CACGAATACT TCTTCCTGAT TGGGCCGCCG 780 CTGCTCATCC CCATGTATTT CCAGTACCAG ATCATCATGA CCATGATCGT CCATAAGAAC 840 TGGGTGGACC TGGCCTGGGC CGTCAGCTAC TACATCCGGT TCTTCATCAC CTACATCCCT 900 TTCTACGGCA TCCTGGGAGC CCTCCTTTTC CTCAACTTCA TCAGGTTCCT GGAGAGCCAC 960 TGGTTTGTGT GGGTCACACA GATGAATCAC ATCGTCATGG AGATTGACCA GGAGGCCTAC 1020 CGTGACTGGT TCAGTAGCCA GCTGACAGCC ACCTGCAACG TGGAGCAGTC CTTCTTCAAC 1080 GACTGGTTCA GTGGACACCT TAACTTCCAG ATTGAGCACC ACCTCTTCCC CACCATGCCC 1140 CGGCACAACT TACACAAGAT CGCCCCGCTG GTGAAGTCTC TATGTGCCAA GCATGGCATT 1200

132

GAATACCAGG AGAAGCCGCT ACTGAGGGCC CTGCTGGACA TCATCAGGTC CCTGAAGAAG 1260 TCTGGGAAGC TGTGGCTGGA CGCCTACCTT CACAAATGAA GCCACAGCCC CCGGGACACC 1320 .GTGGGGAAGG GGTGCAGGTG GGGTGATGGC CAGAGGAATG ATGGGCTTTT GTTCTGAGGG 1380 GTGTCCGAGA GGCTGGTGTA TGCACTGCTC ACGGACCCCA TGTTGGATCT TTCTCCCTTT 1440 CTCCTCTCCT TTTTCTCTTC ACATCTCCCC CATAGCACCC TGCCCTCATG GGACCTGCCC 1500 TCCCTCAGCC GTCAGCCATC AGCCATGGCC CTCCCAGTGC CTCCTAGCCC CTTCTTCCAA 1560 GGAGCAGAGA GGTGGCCACC GGGGGTGGCT CTGTCCTACC TCCACTCTCT GCCCCTAAAG 1620 ATGGGAGGAG ACCAGCGGTC CATGGGTCTG GCCTGTGAGT CTCCCCTTGC AGCCTGGTCA 1680 CTAGGCATCA CCCCCGCTTT GGTTCTTCAG ATGCTCTTGG GGTTCATAGG GGCAGGTCCT 1740 AGTCGGGCAG GGCCCCTGAC CCTCCCGGCC TGGCTTCACT CTCCCTGACG GCTGCCATTG 1800 GTCCACCCTT TCATAGAGAG GCCTGCTTTG TTACAAAGCT CGGGTCTCCC TCCTGCAGCT 1860 CGGTTAAGTA CCCGAGGCCT CTCTTAAGAT GTCCAGGGCC CCAGGCCCGC GGGCACAGCC 1920's AGCCCAAACC TTGGGCCCTG GAAGAGTCCT CCACCCCATC ACTAGAGTGC TCTGACCCTG 1980 GGCTTTCACG GGCCCCATTC CACCGCCTCC CCAACTTGAG CCTGTGACCT TGGGACCAAA 2040 GGGGGAGTCC CTCGTCTCTT GTGACTCAGC AGAGGCAGTG GCCACGTTCA GGGAGGGGCC 2100 GGCTGGCCTG GAGGCTCAGC CCACCCTCCA GCTTTTCCTC AGGGTGTCCT GAGGTCCAAG 2160 ATTCTGGAGC AATCTGACCC TTCTCCAAAG GCTCTGTTAT CAGCTGGGCA GTGCCAGCCA 2220 ATCCCTGGCC ATTTGGCCCC AGGGGACGTG GGCCCTG 2257

(2) Information about SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 411 amino acids (B) TYPE: amino acid (O) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear ín) MOLECULAR TYPE: amino acid (translation contigu 2692004) (xi) Sequence description: SEQ ID NO: 34:

• · • ·

133

His Ala Asp Arg Arg Arg Glu Ile Leu Ala Lys Tyr For Glu Ile 1 5 10 15 Dec Lys Ser Leu Met Lys For Asp For Asn Leu Ile Trp Ile Ile Ile 20 May 25 30 Met Met Wall Leu Thr Gin Leu Gly Ala Phe Tyr Ile Wall Lys Asp 35 40 45 Leu Asp Trp Lys Trp Wall Ile Phe Gly Ala Tyr Ala Phe Gly Ser 50 55 60 Cys Ile Asn His Ser Met Thr Leu Ala Ile His Glu Ile Ala His 65 70 75 Asn Ala Ala Phe Gly Asn Cys Lys Ala Met Trp Asn Arg Trp Phe 80 85 90 Gly Met Phe Ala Asn Leu For Ile Gly Ile For Tyr Ser Ile Ser 95 100 ALIGN! 105 Phe Lys Arg Tyr His Met Asp His His Arg Tyr Leu Gly Ala Asp 110 115 120 Gly Wall Asp Wall Asp Ile For Thr Asp Phe Glu Gly Trp Phe Phe 125 130 135 Cys Thr Ala Phe Arg Lys Phe Ile Trp Wall Ile Leu Gin For Leu 140 145 150 Phe Tyr Ala Phe Arg For Leu Phe Ile Asn For Lys For Ile Thr 155 160 165 Tyr Leu Glu Wall Ile Asn Thr Wall Ala Gin Wall Thr Phe Asp Ile 170 175 180 Leu Ile Tyr Tyr Phe Leu Gly Ile Lys Ser Leu Wall Tyr Met Leu 185 190 195 Ala Ala Ser Leu Leu Gly Leu Gly Leu His For Ile Ser Gly His 200 205 210 Phe Ile Ala Glu His Tyr Met Phe Leu Lys Gly His Glu Thr Tyr 215 220 225 Ser Tyr Tyr Gly For Leu Asn Leu Leu Thr Phe Asn Wall Gly Tyr 230 235 240 His Asn Glu His His Asp Phe For Asn Ile For Gly Lys Ser Leu 245 250 255 For Leu Wall Arg Lys Ile Ala Ala Glu Tyr Tyr Asp Asn Leu For 260 265 270 His Tyr Asn Ser Trp Ile Lys Wall Leu Tyr Asp Phe Wall Met Asp 275 280 285 Asp Thr Ile Ser For Tyr Ser Arg Met Lys Arg His Gin Lys Gly 290 295 300 Glu Met Wall Leu Glu ★ * ★ Ile Ser Leu Wall For Lys Gly Phe Phe 305 310 315 Ser Lys Thr Leu Asp Asp Lys Met Glu Phe Leu His Tyr ★ * * Thr 320 325 330 ★ * ★ Asp Gin * ★ ★ Cys Ser Glu Ala For Leu Ala Gin Phe Gin Ser 335 340 345 Lys Ser Ser Wall Ile For Arg Ser Glu Ser Gly Phe * * * Thr Wall 350 355 360 Ser Leu Thr Leu Tyr Cys Ser Wall Ser Leu Thr Gly Asn Leu * * * 365 370 375 Leu Wall Tyr Tyr Arg His ★ ★ ★ Gly Cys Phe Thr His Wall Cys His 380 385 390 Phe Ile Ser Ile Ser Phe Lys Lys Leu Leu Lys Ser Tyr Phe Ala 400 405 410

Arg ·· • 0 (2) Information about SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 218 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: amino acid (translation of contig

2153526)

(xi) Description sequence: SEQ ID NO: 35: Tyr 1 Leu Leu Arg Pro 5 Leu Leu Pro His Leu Cys Ala Thr Ile Gly Ala Glu Ser Phe Leu Gly Leu Phe Phe Ile Val Arg Phe Leu 30 Ser Asn Trp Phe Val Val Thr Gin Met Asn His Ile Pro Met His Ile Asp His Asp 50 Arg Asn Met Asp Trp Val Ser Thr Gin Leu Gin Ala Thr Cys Asn Val His Lys Ser Ala Phe Asn 71 Asp Trp Phe Ser Gly His Leu Asn 80 Phe Gin Ile Glu His His Leu Phe Pro Thr Met For Arg His Asn Tyr His Lys Val Ala Pro Leu Val Gin Ser Leu Cys Ala Lys His Gly Ile Glu Tyr Gin Ser Lys 116 For Leu Leu 120 Ser Ala Phe Ala Asp Ile Ile His Ser Leu Lys Glu 129 Ser Gly Gin Leu Trp Leu Asp Ala Tyr Leu His Gin Gin Gin 145 For Pro Cys 150 For Val Trp Lys Lys Arg Arg Lys Thr Leu Glu Pro 160 Arg Gin Arg Gly Ala *** Gly Thr Met Pro Leu *** Phe Asn Thr 175 Gin Arg Gly Leu Gly Leu Gly Thr *** Ser Ley *** Leu Lys Leu 190 Leu Pro Phe 195 Ile Glu Phe *** Val Pro For Gin 200 Arg Arg 215 Phe ★ Asp Glu Gly Ala For Lys Trp Gly 205 Val Asp Thr

• ·

• · · • 4 · · · · • · · • · · · · 135 • · · • · · · · · • · ··· ··· • · ·

(2) Information about SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 86 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: amino acid (translation of contig 3506132)

(xi) Description sequence: SEQ ID NO: 2 56: Val Phe Tyr Phe Gly Asn Gly Trp Ile For Thr Leu Ile Thr Ala 1 5 10 15 Dec Phe Val Leu Ala Thr Ser Gin Ala Gin Ala Gly Trp Leu Gin His 20 May 25 30 Asp Tyr Gly His Leu Ser Val Tyr Arg Lys For Lys Trp Asn His 35 40 45 Leu Val His Lys Phe Val Ile Gly His Gly Ala Leu Lys Ser Ala 50 55 60 Asn Trp Trp Asn His Arg His Phe Gin His His Ala Lys Pro Asn 65 70 75 Leu Gly Glu Trp Gin Pro Ile Glu Tyr Gly Lys Xxx 80 85 (2) Information about SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 306 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single-chain (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: amino acid (translation contig 3854933) (xi) Sequence description: SEQ ID NO: 37:

Gin Gly Thr Pro Arg Thr Phe Thr Trp Asp Glu Val Ala Gin 1 5 10

Arg Ser Gly Cys Glu Glu Arg Trp Leu Val Ile Asp

Tvr Asn Ile Ser Glu Phr Thr Arg His Pro Gly Gly Ser Arg 35 40 45 y _a i_ χχθ ser His

Ala Phe His Ile Asn Lys Gly Leu Lys Lys Tyr Met Asn Ser 65 70 75

Leu Leu Ile Glu Glu Pro Glu Pro Glu Pro Glu Pro • ·

80 85 90 Thr Lys Asn Lys Glu Leu Thr Asp Glu Phe Arg Glu Leu Arg Ala 95 100 ALIGN! 105 Thr Wall Glu Arg Met Gly Leu Met Lys Ala Asn His Wall Phe Phe 110 115 120 Leu Leu Tyr Leu Leu His Ile Leu Leu Leu Asp Gly Ala Ala Trp 125 130 135 Leu Thr Leu Trp Wall Phe Gly Thr Ser Phe Leu For Phe Leu Leu 140 145 150 Cys Ala Wall Leu Leu Ser Ala Wall Gin Ala Gin Ala Gly Trp Leu 155 160 165 Gin His Asp Phe Gly His Leu Ser Wall Phe Ser Thr Ser Lys Trp 170 175 180 Asn His Leu Leu His His Phe Wall Ile Gly His Leu Lys Gly Ala 185 190 195 For Ala Ser Trp Trp Asn His Met His Phe Gin His His Ala Lys 200 205 210 For Asn Cys Phe Arg Lys Asp For Asp Ile Asn Met His For Phe 215 220 225 Phe Phe Ala Leu Gly Lys Xle Leu Ser Wall Glu Leu Gly Lys Gin 230 235 240 Lys Lys Lys Tyr Met For Tyr Asn His Gin His Xxx Tyr Phe Phe 245 250 255 Leu Ile Gly For For Ala Leu Leu For Leu Tyr Phe Gin Trp Tyr 260 265 270 ile Phe Tyr Phe Wall Ile Gin Arg Lys Lys Trp Wall Asp Leu Ala 275 280 285 Trp Ile Ser Lys Gin Glu Tyr Asp Glu Ala Gly Leu For Leu Ser 290 295 300 Thr Ala Asn Ala Ser Lys 305

(2) Information about SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 566 amino acids (E) TYPE: amino acid (F) CHAIN TYPE: single chain (G) TOPOLOGY: linear (ii) MOLECULAR TYPE: amino acid (translation contig 2511785) (xi) Sequence description: SEQ ID NO: 38:

His Leu Lys Gla Ala Ser Ala Asn Trp Asp His Arg His Phe 1 5 10 15

Gin His His Lys Pro Asn Ile Phe His Lys Asp Pro Asp Val

25 30

Asn Met Leu Gly Glu Trp Gin Pro Ile Glu

40 45

Tyr Gly Lys Lys Lys Lys Lys Tyr Leu For Tyr Asn His Gin His

55 60

Glu Tvr Phe Phe Phu Leu Ile Gly Pro Leu Leu Ile Pro Met Tyr

70 75

Phe Gin Tyr Gin Ile Ile Met Thr Ile Val His Lys Asn Trp

85 90

Val Asp Leu Ala Trp Ala Val Ser Tyr Tyr Ile Arg Phe Phe Ile

100 105

Ile Pro Phe Tyr

110 115 120

Asn Phe Ile Arg, Phe Leu Glu Ser His Php Phe Val Php Val Thr

126 130 135

Gin Met Asn His Gle Glu Gle Ile Asp Gin Glu Ala Tyr Arg

140 145 150

137

Asp Trp Phe Ser Ser Gin Leu Thr Cys Asn Val Glu Gin 165 155 160 Ser Phe Phe Asn Asp Trp Phe Ser Gly His Leu Asn Phe Gin Ile 170 175 180 Glu His His Leu Phe For Thr Met For Arg His Asn Leu His Lys 185 190 195 Ile Ala For Leu Wall Lys Ser Leu Cys Ala Lys His Gly Ile Glu 200 205 210 Tyr Gin Glu Lys For Leu Leu Arg Ala Leu Leu Asp Ile Ile Arg 215 220 225 Ser Leu Lys Lys Ser Gly Lys Leu Trp Leu Asp Ala Tyr Leu His 230 235 240 Lys * ★ ★ Ser His Ser For Arg Asp Thr Wall Gly Lys Gly Cys Arg 245 250 255 Trp Gly Asp Gly Gin Arg Asn Asp Gly Leu Leu Phe ir * * Gly Wall 260 265 270 Ser Glu Arg Leu Wall Tyr Ala Leu Leu Thr Asp For Met Leu Asp 275 280 285 Leu Ser For Phe Leu Leu Ser Phe Phe Ser Ser His Leu For His 290 295 300 Ser Thr Leu For Ser Trp Asp Leu For Ser Leu Ser Arg Gin For 305 310 315 Ser Ala Met Ala Leu For Wall For For Ser For Phe Phe Gin Gly 320 325 330 Ala Glu Arg Trp For For Gly Wall Ala Leu Ser Tyr Leu His Ser 335 340 345 Leu For Leu Lys Met Gly Gly Asp Gin Arg Ser Met Gly Leu Ala 350 355 360 Cys Glu Ser For Leu Ala Ala Trp Ser Leu Gly Ile Thr For Ala 365 370 375 Leu Wall Leu Gin Met Leu Leu Gly Phe Ile Gly Ala Gly For Ser 380 385 390 Arg Ala Gly For Leu Thr Leu For Ala Trp Leu His Ser For ♦ * * 400 405 410 Arg Leu For Leu Wall His For Phe Ile Glu Arg For Ala Leu Leu 415 420 425 Gin Ser Ser Gly Leu For For Ala Ala Arg Leu Ser Thr Arg Gly 430 435 440 Leu Ser ★ 1e Asp Wall Gin Gly For Arg For Ala Gly Thr Ala Ser 445 450 455 For Asn Leu Gly For Trp Lys Ser For For For His His * * * Ser 460 465 470 Ala Leu Thr Leu Gly Phe His Gly For His Ser Thr Ala Ser For 475 480 485 Thr ★ ★ jt Ala Cys Asp Leu Gly Thr Lys Gly Gly Wall For Arg Leu 490 495 500 Leu * * * Leu Ser Arg Gly Ser Gly His Wall Gin Gly Gly Ala Gly 505 510 515 Trp For Gly Gly Ser Ala His For For Ala Phe For Gin Gly Wall 520 525 530 Leu Arg Ser Lys Ile Leu Glu Gin Ser Asp For Ser For Lys Ala 535 540 545 Leu Leu Ser Ala Gly Gin Cys Gin For Ile For Gly His Leu Ala 550 555 560 For Gly Asp Wall Gly For Xxx 565

(2) Information about SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 619 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: one-string (D) TOPOLOGY: linear • · (ii) MOLECULAR TYPE: amino acid (translation of contigu 2535

(xi ) Description S ¹ equivalence: i 3EQ ID NO: 39: Wall 1 Phe Tyr Phe Gly Asn Gly Trp Ile For 10 Thr Leu Ile Thr Ala 14 Phe Wall Leu Ala Thr Ser Gin Ala Gin Ala 25 Gly Trp Leu Gin His Asp Tyr Gly His Leu Ser Val Arg Lys 40 For Lys Trp Asn His 45 Leu Wall His Lys Phe Val Gle His Leu 55 Lys Gly Ala Ser Ala 60 Asn Trp Trp Asn His Arg His Phe Gin His 70 His Ala Lys Pro Asn Ile Phe His Lys Asp 80 For Asp Val Asn Met 85 Leu His Val Phe Val Leu Gly Glu Trp Gin For Ile Glu Tyr Gly 100 ALIGN! Lys Lys Lys Leu Lys Tyr Leu For Tyr Asn 110 His Gin His Glu Tyr 115 Phe Phe Leu Ile Gly 119 For For Leu Leu Ile For Met Tyr Phe Gin 130 Tyr Gin Ile Ile Met Thr Met Ile Val His 140 Lys Asn Trp Wall Asp 145 Leu Ala Trp Ala Val Ser Tyr Tyr Ile Arg Phe Phe Ile Thr Tyr 160 Ile Pro Phe Tyr Gly 165 Ile Leu Gly Ala Leu Phu Leu Asn Phe 175 Ile Arg Phe Leu Glu Ser His Trp Phe Val 185 Val Thr Gin Met 190 Asn His Ile Val Met 195 Glu Ile Asp Gin Glu 200 Ala Tyr Arg Asp Trp 205 Phe Ser Ser Gin Leu Thr Ala Thr Cys Asn 215 Val Glu Ser Phe 220 Phe Asn Asp Trp Phe 225 Ser Gly His Leu Asn Phe Gin Ile Glu His 235 His Leu Phe Pro Thr 240 Met For Arg His Asn Leu His Lys Ile Ala 250 For Leu Val Lys Ser 255 Leu Cys Ala Lys His 260 Gly Ile Glu Tyr Gin 265 Glu Lys Pro Leu Leu 270 Arg Ala Leu Leu Asp 275 Arg Ser Leu 280 Lys Lys Ser Gly Lys Leu Trp Leu Asp Ala 290 Tyr Leu His Lys ★ ★ ★ 295 Ser His Ser Pro Arg 300 Asp Thr Val Gly Lys Gly Cys Arg Trp Gly 310 Asp Gly Gin Arg Asn 315 Asp Gly Leu Leu Phe 320 *** Gly Val Ser Glu 325 Arg Leu Val Tyr Ala 330 Leu Leu Thr Asp Pro 335 Met Leu Asp Leu Ser 340 For Phe Leu Leu Ser 345 Phe Phe Ser Ser His 350 Leu For His Ser Thr 355 Leu For Ser Trp Asp 360 Leu For Ser Leu Ser 365 Arg Gin Pro Ser Ala 370 Met Ala Leu For Val 375 For For Ser Pro Phe 380 Phe Gin Ala Glu 385 Arg Trp Pro Gly 390 Wall Ala Leu Ser Tyr 400 Leu His Ser Leu For 405 Leu Lys Met Gly Gly 410 Asp Gin Arg Ser Met Gly Leu Ala Cys Glu 420 Ser For Leu Ala Ala 425 Trp Ser Leu Gly Ile 430 Thr Pro Ala Leu Wall 435 Leu Gin Met Leu Leu 440 Gly Phe Ile Gly Ala 444 Gly Pro Ser Arg Ala 450 Gly For Leu Thr Leu 455

«· • ·

139

Pro Ala Trp Leu His 460 Ser For ★ ★ ★ Arg Leu 4 65 For Leu Val His For 470 Phe Ile Glu Arg For Ala Leu Leu Gin Ser Ser Gly Leu For For 475 480 485 Ala Ala Arg Leu Ser Thr Arg Gly Leu Ser * * * Asp Wall Gin Gly 490 495 500 For Arg For Ala Gly Thr Ala Ser For Asn Leu Gly For Trp Lys 505 510 515 Ser For For For His His *** Ser Ala Leu Thr Leu Gly Phe His 520 525 530 Gly For His Ser Thr Ala Ser For Thr ★ ** Ala Cys Asp Leu Gly 535 540 545 Thr Lys Gly Gly Wall For Arg Leu Leu ★ ★ ★ Leu Ser Arg Gly Ser 550 555 560 Gly His Wall Gin Gly Gly Ala Gly Trp For Gly Gly Ser Ala His 565 570 575 For For Ala Phe For Gin Gly Wall Leu Arg Ser Lys Ile Leu Glu 580 585 590 Gin Ser Asp For Ser For Lys Ala Leu Leu Ser Ala Gly Gin Cys 595 600 605 Gin For Ile For Gly His Leu Ala For Gly Asp Wall Gly For Xxx 610 615 620

(2) Information about SEQ ID NO: 40:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH : 757 amino acids (B) TYPE: amino acid (C) SPECIES CHAINS: single chain (D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: amino acid (translation of contig 253538a) (xi) Description of sequence: SEQ ID NO: 40:

Gin Gly For Thr For Arg Tyr Phe Thr Trp Asp Glu Wall Ala Gin 1 5 10 15 Dec Arg Ser Gly Cys Glu Glu Arg Trp Leu Wall Ile Asp Arg Lys Wall 20 May 25 30 Tyr Asn Ile Ser Glu Phe Thr Arg Arg His For Gly Gly Ser Arg 35 40 45 Wall Ile Ser His Tyr Ala Gly Gin Asp Ala Thr Asp For Phe Wall 50 55 60 Ala Phe His Ile Asn Lys Gly Leu Wall Lys Lys Tyr Met Asn Ser 65 70 75 Leu Leu Ile Gly Glu Leu Ser For Glu Gin For Ser Phe Glu For 80 85 90 Thr Lys Asn Lys Glu Leu Thr Asp Glu Phe Arg Glu Leu Arg Ala 95 100 ALIGN! 105 Thr Wall Glu Arg Met Gly Leu Met Lys Ala Asn His Wall Phe Phe 110 115 120 Leu Leu Tyr Leu Leu His Ile Leu Leu Leu Asp Gly Ala Ala Trp 125 130 135 Leu Thr Leu Trp Wall Phe Gly Thr Ser Phe Leu For Phe Leu Leu 140 145 150 Cys Ala Wall Leu Leu Ser Ala Wall Gin Gin Ala Gin Ala Gly Trp 155 160 165 Leu Gin His Asp Tyr Gly His Leu Ser Wall Tyr Arg Lys For Lys 170 175 180 Trp Asn His Leu Wall His Lys Phe Wall Ile Gly His Leu Lys Gly 185 190 195 Ala Ser Ala Asn Trp Trp Asn His Arg His Phe Gin His His Ala 200 205 210

140 •

Lys For Asn Ile Phe 215 His Lys Asp Pro Asp 220 Wall Asn Met Leu His 225 Wall Phe Wall Leu Gly Glu Trp Gin For Ile Glu Tyr Gly Lys Lys 230 235 240 Lys Leu Lys Tyr Leu For Tyr Asn His Gin His Glu Tyr Phe Phe 245 250 255 Leu Ile Gly For For Leu Leu Ile For Met Tyr Phe Gin Tyr Gin 260 265 270 Ile Ile Met Thr Met Ile Wall His Lys Asn Trp Wall Asp Leu Ala 275 280 285 Trp Ala Wall Ser Tyr Tyr Ile Arg Phe Phe Ile Thr Tyr Ile For 290 295 300 Phe Tyr Gly Ile Leu Gly Ala Leu Leu Phe Leu Asn Phe Ile Arg 305 310 315 Phe Leu Glu Ser His Trp Phe Wall Trp Wall Thr Gin Met Asn His 320 325 330 Ile Wall Met Glu Ile Asp Gin Glu Ala Tyr Arg Asp Trp Phe Ser 335 340 345 Ser Gin Leu Thr Ala Thr Cys Asn Wall Glu Gin Ser Phe Phe Asn 350 355 360 Asp Trp Phe Ser Gly His Leu Asn Phe Gin Ile Glu His His Leu 365 370 375 Phe For Thr Met For Arg His Asn Leu His Lys Ile Ala For Leu 380 385 390 Wall Lys Ser Leu Cys Ala Lys His Gly Ile Glu Tyr Gin Glu Lys 400 405 410 For Leu Leu Arg Ala Leu Leu Asp Ile Ile Arg Ser Leu Lys Lys 415 420 425 Ser Gly Lys Leu Trp Leu Asp Ala Tyr Leu His Lys ★ i ★ Ser His 430 435 440 Ser For Arg Asp Thr Wall Gly Lys Gly Cys Arg Trp Gly Asp Gly 445 450 455 Gin Arg Asn Asp Gly Leu Leu Phe * * * Gly Wall Ser Glu Arg Leu 460 4 65 470 Wall Tyr Ala Leu Leu Thr Asp For Met Leu Asp Leu Ser For Phe 475 480 485 Leu Leu Ser Phe Phe Ser Ser His Leu For His Ser Thr Leu For 490 495 500 Ser Trp Asp Leu For Ser Leu Ser Arg Gin For Ser Ala Met Ala 505 510 515 Leu For Wall For For Ser For Phe Phe Gin Gly Ala Glu Arg Trp 520 525 530 For For Gly Wall Ala Leu Ser Tyr Leu His Ser Leu For Leu Lys 535 540 545 Met Gly Gly Asp Gin Arg Ser Met Gly Leu Ala Cys Glu Ser For 550 555 560 Leu Ala Ala Trp Ser Leu Gly Ile Thr For Ala Leu Wall Leu Gin 565 570 575 Met Leu Leu Gly Phe Ile Gly Ala Gly For Ser Arg Ala Gly For 580 585 590 Leu Thr Leu For Ala Trp Leu His Ser For * * * Arg Leu For Leu 595 600 605 Wall His For Phe Ile Glu Arg For Ala Leu Leu Gin Ser Ser Gly 610 615 620 Leu For For Ala Ala Arg Leu Ser Thr Arg Gly Leu Ser * * * Asp 625 630 635 Wall Gin Gly Fro Arg For Ala Gly Thr Ala Ser For Asn Leu Gly 640 645 650 For Trp Lys Ser For For For His His ★ ★ ♦ Ser Ala Leu Thr Leu 655 660 665 Gly Phe His Gly For His Ser Thr Ala Ser For Thr *** Ala Cys 670 675 680 Asp Leu Gly Thr Lys Gly Gly Wall For Arg Leu Leu ★ * * Leu Ser 685 690 695 Arg Gly Ser Gly His Wall Gin Gly Gly Ala Gly Trp For Gly Gly 700 705 710

• · ·

141

Ser Ala His For For 715 Ala Phe For Gin Gly Val 720 Leu Arg Ser Lys 725 Ile Leu Glu Gin Ser Asp For Ser For Lys Ala Leu Leu Ser Ala 730 735 740 Gly Gin Cys Gin For Ile For Gly His Leu Ala For Gly Asp Wall 745 750 755

Gly Pro Xxx

Claims (14)

PATENT CLAIMS 142 An isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3. The polypeptide encoded by the nucleic sequence of claim 1. A purified and isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. An isolated nucleic acid encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. An isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide that desaturates a fatty acid molecule at the sixth or twelfth carbon position from the carboxyl terminus of said polypeptides, wherein said nucleotide sequence has an average A / T content of less than about 60%. 6. Isolated nucleic acid according to claim 5, where listed nuclear the acid was obtained of mushrooms. 7. Isolated nucleic acid according to claim 6, where mentioned the sponge is genus Mortierella. 8. Isolated nucleic acid according to claim 7, where listed the fungus is species Mortierella alpina. 9. Isolated nucleic acid where nucleotide sequence said nucleic acid being set forth in SEQ ID NO: 1 or SEQ ID NO: 3. An isolated or purified polypeptide that desaturates a fatty acid molecule at the sixth or twelfth carbon position from the carboxyl terminus of said polypeptide which is a eukaryotic polypeptide or is derived from a eukaryotic polypeptide. The isolated and purified eukaryotic polypeptide of claim 10, which is derived from fungi. • · 143 A nucleic acid comprising a nucleotide sequence of a fungus that is substantially identical to the sequence of at least 50 nucleotides in SEQ ID NO: 1 or SEQ ID NO: 3 or is complementary to the sequence of at least 50 nucleotides in SEQ ID NO: 1 or in SEQ ID NO: 3. An isolated nucleic acid having a nucleotide sequence having at least about 50% homology to SEQ ID NO: 1 or SEQ ID NO: 3. An isolated nucleic acid having a nucleotide sequence having at least about 50% homology to the sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4. The nucleic acid of claim 14, wherein said amino acid sequence comprised in SEQ ID NO 2 is selected from the group consisting of amino acid residues 50-53, 39-43, 172-176, 204-213 and 390-402. 16. A nucleic acid construct comprising the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 associated with a heterogeneous nucleic acid. 17. A nucleic acid construct comprising the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 operably linked to an expression control sequence that is functional in a mincrobial cell. 18. Nucleic acid construction according to claim 17, where mentioned the microbial cell is a yeast cell. 19 Dec Nucleic acid construction according to claim 17, where listed the nucleotide sequence is derived from hub. 20 May Nucleic acid construction according to claim 19, where listed the fungus is of the genus Mortierella. 21. Nucleic acid construction according to claim 20, where listed the fungus is of the species Mortierella alpina. 22. A nucleic acid construct comprising a fungal nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that corresponds to or is 144 complementary to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, wherein said nucleic acid is operably linked to an expression control sequence that is functional in a microbial cell, wherein said nucleotide sequence encodes a functionally active polypeptide that desaturates the molecule fatty acids at the sixth and twelfth carbon positions from the carboxyl terminus of the fatty acid molecule. A nucleic acid construct comprising a nucleotide sequence having an A / T content of less than about 60% and encoding a functionally active δδ-desaturase with an amino acid sequence corresponding to or complementary to all or part of the amino acid sequence shown in SEQ ID NO: 2, operably said nucleotide sequence being linked to the sequence controlling transcription that is functional in the yeast cell. A nucleic acid construct comprising a fungal nucleotide sequence that encodes a functionally active A12-desaturase with an amino acid sequence that matches or is complementary to all or part of the amino acid sequence shown in SEQ ID NO: 4, said nucleotide sequence being operably linked to a transcriptional control sequence, which is functional in a yeast cell. A recombinant yeast cell comprising the nucleic acid construct of claim 23 or 24. yeast according to claim 25, wherein Recombinant cell indicating Saccharomyces. A recombinant cell is a cell of yeast comprising at least one copy of a vector comprising a fungal nucleotide sequence that encodes a polypeptide that converts 18: 2 fatty acids to 18: 3 fatty acids or 18: 3 fatty acids to fatty acids. 145 18: 4, wherein said yeast cell or precursor thereof is transformed with said vector to produce said recombinant yeast cell, and wherein said nucleotide sequence is operably linked to an expression control sequence that is functional in said recombinant yeast cell. The recombinant yeast cell of claim 27, wherein the cell sequence is that the fungus is a nucleotide yeast according to said sequence of claim 28, the cell is a nucleotide cell. Mortierella. Recombinant designating Saccharomyces. The microbial cell of claim 27, wherein said expression control sequence is located in said expression vector. A method of producing GLA in a culture comprising culturing a yeast culture comprising large yeast comprising: a plurality of recombinant yeast cells, wherein said yeast cells or precursors thereof are transformed with a vector comprising a fungal DNA encoding a polypeptide that converts LA to GLA, said DNA is operably linked to an expression control sequence that is functional in said yeast cells under conditions whereby DNA is expressed, thereby producing GLA from LA in said yeast culture. The method of claim 31, wherein said fungal DNA is Mortierella DNA and said polypeptide is 66-desaturase. The method of claim 32, wherein Mortierella is a species of Mortierella alpina. The method of claim 31 wherein said LA is supplied exogenously The method of claim 31, characterized in that said conditions can be induced. • · « 146 36. A method of producing stearidonic acid in a yeast culture, the method comprising: culturing a yeast culture comprising a plurality of recombinant yeast cells, wherein said yeast cells or precursors thereof are transformed with a vector comprising a fungal DNA encoding a polypeptide that converts α-linolenic acid to stearidonic acid, said DNA operably linked to an expression control sequence that is functional in said yeast cells under conditions whereby the DNA is expressed, thereby producing said α-linolenic acid in said yeast culture. The method of claim 36, wherein said fungal DNA is Mortierella DNA said polypeptide is β-desaturase. 38. The method of claim 37 wherein Mortierella is a species of Mortierella alpina. stearidonic acid characterized by a The method of claim 36 40. The method, wherein said α-linolenic acid is supplied exogenously. according to claim 36, characterized in that said conditions can be induced. A method of producing linolenic acid in a yeast culture, comprising: culturing a yeast culture comprising a plurality of said recombinant yeast cells, wherein the yeast cells or their precursors have been transformed with a vector comprising a fungal DNA encoding a polypeptide that converts oleic acid to linolenic acid, said DNA operably linked to an expression control sequence that is functional yeast cells under DNA expression conditions, thereby producing linolenic acid from oleic acid in said yeast culture. 147 • · · · < 42. The method of claim 41, wherein said fungal DNA is Mortierella DNA and said polypeptide is 1212-desaturase. 43. Way according to claim 42, mark i c i se t and m that Mortierella is a species Mortierella alpina • 44. Way according to Claim 41 characterized c í se t and m that these conditions are can trigger. 45. Isolated or purified polypeptide which desaturates molecule fatty acid in position of the 12th carbon from desaturate carbon from the carboxyl terminus of said polypeptides, wherein said polypeptide is a fungal polypeptide or is derived from fungal polypeptides. The isolated or purified polypeptide of claim 46 which is an A12-desaturase of Mortierella alpina. An isolated or purified polypeptide having a fatty acid molecule at the sixth carboxyl terminus of said polypeptide, wherein said polypeptide is a fungal polypeptide or is derived from a fungal polypeptide. The isolated or purified polypeptide of claim 48 which is β-desaturase. The isolated nucleic acid encoding the polypeptide of claim 47 or 49. The nucleic acid construct of claim 23, wherein said portion of the amino acid sequence shown in SEQ ID NO: 2 comprises amino acids 1 to 457. A host cell comprising the nucleic acid construct of any one of claims 22 to 24. A host cell comprising a vector comprising a nucleic acid that encodes a fatty acid desaturase derived from Mortierella alpina, characterized in that said desaturase has an amino acid sequence represented by SEQ ID NO. 148 NO: 2, wherein said nucleotide sequence is operably linked to a promoter. 53. 54. 55. 56. 57. Host cell by Claim 52 confesses č u j i c i by as mentioned above host the cell is eukaryotic cell. Host cell by Claim 53 confesses č u j i c i by as mentioned above eukaryotic cell with chose that groups comprising a mammalian cell, a plant cell, an insect cell, cell Host fungi, a bird cell and a cell according to claim cell 54, eyelashes. marked at .mu.Ci by Host is a cell of fungi. a cell according to claim 21, marked at .mu.Ci by wherein said promoter is into listed host cells delivered exogenously. Process for producing stearidonic acid in culture eukaryotic cells č u j ící se by comprising: culturing a culture of eukaryotic cells comprising a plurality of recombinant eukaryotic cells, wherein said recombinant eukaryotic cells or precursors thereof are transformed with a vector comprising a fungal DNA encoding a polypeptide that converts alinolenic acid to stearidonic acid, said DNA operably linked to an expression control sequence which is functional in said yeast cells under conditions whereby DNA is expressed, thereby producing stearidonic acid from alinolenic acid in said culture of eukaryotic cells. A method of producing linolenic acid in a culture of eukaryotic cells, comprising: culturing a culture of eukaryotic cells comprising a plurality of recombinant eukaryotic cells, wherein said recombinant eukaryotic cells or precursors thereof have been transformed with a vector comprising a fungal DNA encoding an acid converting polypeptide 58. Wherein said DNA is operably linked to an expression control sequence that is functional in said recombinant eukaryotic cells under conditions whereby DNA is expressed, thereby producing linolenic acid from oleic acid in said culture of eukaryotic cells. The method of claim 57 or 58, wherein said eukaryotic cells are selected from the group consisting of mammalian cells, plant cells, insect cells, fungal cells, avian cells and algae cells. The method of claim 59, wherein said fungal cells are yeast cells of the genus Saccharomyces. Recombinant yeast cell containing: (1) at least one nucleic acid construct according to claim 23 or 24; or (2) at least one nucleic acid construct according to claim 23 and at least one nucleic acid construct according to claim 24. A recombinant yeast cell comprising at least one nucleic acid construct comprising a nucleotide sequence that encodes a functionally active -6-desaturase having an amino acid sequence that matches or is complementary to all or part of the amino acid sequence shown in SEQ ID NO: 2 and at least one nucleic acid construct comprising a nucleotide sequence that encodes a functionally active Δ12-desaturase with an amino acid sequence that matches or is complementary to all or part of the amino acid sequence shown in SEQ ID NO: 4, wherein said nucleic acid constructs are operably linked to transcriptional control sequences that are functional in a yeast cell. • · 150 The method is by preparing The GLA comprising culturing the recombinant yeast cell of claim 62 under conditions whereby said nucleotide sequences are expressed, thereby producing GLA in said yeast cell. The way 66, the preparation of a GLA comprising culturing the recombinant yeast cell of claim 61 under conditions wherein said nucleotide sequences are expressed in said nucleic acid constructs, thereby producing GLA in said yeast cell. A method of obtaining a modified long-chain poly-unsaturated fatty acid biosynthesis comprising the step of activating a plant having cells containing one or more transgenes derived from fungi and algae that encode a transgenic expression product desaturating the fatty acid molecule at the carbon position selected a group comprising sixth and twelfth carbon from the carboxyl terminus of said fatty acid molecule, said one or more transgenes operably linked to an expression control sequence under conditions whereby one or more transgenes are expressed, thereby altering polyunsaturated biosynthesis in said cells long chain fatty acids. Claim 65, characterized in that said polyunsaturated fatty acid is selected from the group consisting of The method is selected by selecting 18: 1099, LA, GLA, SDA and ALA with the long chain. Microbial oil or a fraction thereof, characterized in that it is produced by the method of claim 65. A method of treating or preventing malnutrition, comprising administering said microbial oil of claim 67 to a patient in need of said treatment. · * * 151 69. 70. 71 72. 73. 74. 75. 76. or prevention, in an amount sufficient to effect said treatment or prevention. A pharmaceutical composition comprising said microbial oil or fraction of claim 67 and a pharmaceutically acceptable carrier. Pharmaceutical composition according to a liquid form. Pharmaceutical composition according to characterized caspules or tablets. Pharmaceutical claims 69, being solid or by the composition of claim 69 70, form 69, a food comprising group further comprising at least one nutrient selected from the group consisting of vitamin, mineral, carbohydrate, sugar, amino acid, free fatty acid, phospholipid, antioxidant, and phenolic compound. A nutritional composition comprising said microbial oil or a fraction thereof according to claim 67. A nutritional composition according to claim 73, characterized in that it is selected from infant formulas, food supplements and substitutes. The nutritional composition of claim 74, wherein said infant formula, food supplement and food substitute is in liquid or solid form. Infant formula comprising said microbial oil or a fraction thereof according to claim 67. The infant formula of claim 76, further comprising at least one macronutrient selected from the group consisting of coconut oil, soybean oil, 77. • · 152 canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed non-fat milk, milk whey, soy protein and other protein hydrolysates. The infant formula of claim 77, further comprising at least one vitamin selected from the group consisting of vitamins A, C, D, E and B complex and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodide, selenium and iron. A food supplement comprising microbial oil or a fraction thereof according to claim 67. The food supplement of claim 79 further comprising at least one macronutrient selected from the group consisting of coconut oil, soybean oil, canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed non-fat milk, whey, soy protein and other protein hydrolysates. The dietary supplement of claim 80, further comprising at least one vitamin selected from the group consisting of vitamins A, C, D, E and B complex and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium, and iron. The dietary supplement of claim 79 or 81, which is administered to a human or animal. A food substitute comprising said microbial oil or a fraction thereof according to claim 67. The food substitute of claim 83, further comprising at least one macronutrient 153 selected from the group consisting of coconut oil, soybean oil, canola oil, mono- and diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed nonfat milk, milk whey, soy protein and other protein hydrolysates. 85. The food substitute of claim 84, further comprising at least one vitamin selected from the group consisting of vitamins A, C, D, E, and B complex and at least one mineral selected from the group consisting of calcium, magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium and iron. 86. The food substitute of claim 83 or 85, v y z n and watching with that aplikuj e man or animal. 8 7. Way treatment of the patient suffering from a condition caused insufficient intake or production of polyunsaturated fatty acids, characterized in that said patient is administered said food substitute according to claim 83 or said food supplement according to claim 79 in an amount sufficient to effect said treatment. 88. The method of claim 87 wherein said food substitute or said food supplement is administered enterally or parenterally. 89. A cosmetic composition comprising said microbial oil; a fraction thereof according to claim 67. 90. Cosmetic preparation according to claim 88, ..... emerging by of e se applies surface. 91. Pharmaceutical composition according to claim 69, ..... emerging by of e se aplikuj e human or animal. 154 · · · · · · · · · · · 92. Animal feed, marked u with e by of E contain e said microbial oil or a fraction thereof according to Claim 67 • 93. Way according to claim 20, v y z n a č u j í c and is three m that said sponge i is kind genus Mortierella. 94. Way according to claim 93, v y z n a č u j í c and wherein said fungus is Mortierella alpina. 95. An isolated peptide sequence selected from the group consisting of SEQ ID NO: 34 to SEQ ID NO: 40. 96. An isolated peptide sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, and SEQ ID NO: 26. 97. A method of producing gamma-linolenic acid in a culture of eukaryotic cells, the method comprising: culturing a culture of eukaryotic cells comprising a plurality of recombinant eukaryotic cells, wherein said recombinant eukaryotic cells or precursors thereof are transformed with a vector containing a fungal DNA encoding a polypeptide converting linoleic acid into gamma-linolenic acid, said DNA operably linked to an expression control sequence that is functional in said recombinant eukaryotic cells, under conditions where said DNA is expressed, thereby producing gamma-linolenic acid in said eukaryotic cell culture from linoleic acid. 98. The method of claim 97, wherein said eukaryotic cells are selected from the group consisting of mammalian cells, plant cells, insect cells, fungal cells, avian cells, and algae cells.